Safety assessment of liver-targeted hydrodynamic gene delivery in dogs

Abstract

Evidence in support of safety of a gene delivery procedure is essential toward gene therapy. Previous studies using the hydrodynamics-based procedure primarily focus on gene delivery efficiency or gene function analysis in mice. The current study focuses on an assessment of the safety of computer-controlled and liver-targeted hydrodynamic gene delivery in dogs as the first step toward hydrodynamic gene therapy in clinic. We demonstrate that the impacts of the hydrodynamic procedure were limited in the injected region and the influences were transient. Histological examination and the hepatic microcirculation measurement using reflectance spectrophotometry reveal that the liver-specific impact of the procedure involves a transient expansion of the liver sinusoids. No systemic damage or toxicity was observed. Physiological parameters, including electrocardiogram, heart rate, blood pressure, oxygen saturation, and body temperature, remained in normal ranges during and after hydrodynamic injection. Body weight was also examined to assess the long-term effects of the procedure in animals who underwent 3 hydrodynamic injections in 6 weeks with 2-week time interval in between. Serum biochemistry analysis showed a transient increase in liver enzymes and a few cytokines upon injection. These results demonstrate that image-guided, liver-specific hydrodynamic gene delivery is safe.

Figure 1. Liver-targeted hydrodynamic gene delivery.

(a) Location of the balloon catheter in the hepatic vein. A small amount of contrast medium was injected into the vein to confirm the occlusion of the blood flow and distribution of injected solution during a liver lobe-specific hydrodynamic gene delivery. Black arrows represent the distribution of the contrast medium in the injected liver lobe. (b) Immunohistochemical staining of the liver. Liver samples from control and plasmid DNA injected liver were stained with anti-luciferase antibodies. Scale bar represents 50 µm. White arrow indicates the positively stained hepatocytes. (c) The change of body weight before, after the arrival of the animal to the animal facility. White, gray, and black arrowheads point the hydrodynamic injections in representative 4 dogs. (d–g) Physiological impacts of the sequential, liver-targeted hydrodynamic gene delivery in representative 4 dogs. Physiological parameters before, during, and after the sequential liver-targeted hydrodynamic injection of saline (d), pCMV-Luc (e), pCAG-hAAT (f), or pBS-HCRHP-FIXIA (g). HR, heart rate (white square); SBP, systolic blood pressure (white circle); DBP, diastolic blood pressure (black circle); SpO₂, oxygen saturation (black diamond); BT, body temperature (black square). Black arrows indicate each of the 4 lobe injections.

Figure 2. Electrocardiograms of animals prior to, immediately following, and 5 and 20 min after each of 4 lobe-specific injections.

The electrocardiogram was monitored before, immediately following, 5 min, and 20 min after each of injection of pCMV-Luc to right lateral lobe (a), right medial lobe (b), left medial lobe (c), and left lateral lobe (d) in dog 1 with 20 min of interval time between 2 injections.

Figure 3. Impact of liver-targeted hydrodynamic gene delivery on hepatic sinusoidal structure.

Hematoxylin and eosin staining was performed on the liver sample before (a), immediately following (b), and 3 h (c) after the liver-targeted hydrodynamic gene delivery in rats, and prior to (d), immediately following (e), and 24 h (f) after the liver-targeted hydrodynamic gene delivery in dogs. Scale bar represents 100 µm. (g) Quantitative measurement of hepatic sinusoidal areas. Three different liver sections from 3 rats (n = 9) and 2 dogs (n = 6) were stained and images were captured. Quantitative analysis was performed using ImageJ software (version 1.6.0_20, National Institutes of Health, USA). The values represent mean ± SD. ** p<0.01, *** p<0.001 by ANOVA and Bonferroni's multiple comparison test.

Figure 4. Impact of liver-targeted hydrodynamic gene delivery on hepatic microcirculation.

The total blood volume (IHB, white square) and the blood oxygenation (ISO₂, black diamond) in regional hepatic tissue under normal conditions (a), prior to, during, and after the liver-targeted hydrodynamic saline injection in 10 sec (b), 15 sec (c), and 20 sec (d) to rat livers.

Figure 5. Impact of the procedure on serum biochemistry.

Blood samples were collected from the cephalic or saphenous veins of dogs before (time  = 0), and 2, 4, 24, 96 h after the first hydrodynamic injection of saline (white diamond), pCAG-hAAT (white square), pBS-HCRHP-FIXIA (white triangle), pCMV-Luc (black cross) or slow drip infusion of the same volume of saline from peripheral vein within 1 h (white circle) or 2 h (black asterisk). Concentrations of aspartate aminotransferase (AST) (a), alanine aminotransferase (ALT) (b), lactate dehydrogenase (LDH) (c), creatinine (Cre) (d), albumin (Alb) (e), and hematocrit value (f). The values represent mean for hydrodynamic injection groups (n = 2 for each group).

Figure 6. Impact of the procedure on serum cytokine levels.

Blood samples were collected from the cephalic or saphenous veins of dogs before (time  = 0), and 2, 24, 48, 96 h after the first hydrodynamic injection of saline (white diamond), pCAG-hAAT (white square), pBS-HCRHP-FIXIA (white triangle), pCMV-Luc (black cross) or slow drip infusion of the same volume of saline from peripheral vein within 1 h (white circle) or 2 h (black asterisk). Concentrations of TNF-α (a), IL-10 (b), MCP-1 (c), Canine KC (d), IL-6 (e), IFN-γ (f), IL-8 (g), IL-18 (h), IL-4 (i). The values represent mean (n = 2 for each group).