Tsutomu Kanefuji

Safety Assessment of Liver-Targeted Hydrodynamic Gene Delivery in Dogs

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.

Novel electric power-driven hydrodynamic injection system for gene delivery: safety and efficacy of human factor IX delivery in rats.

The development of a safe and reproducible gene delivery system is an essential step toward the clinical application of the hydrodynamic gene delivery (HGD) method. For this purpose, we have developed a novel electric power-driven injection system called the HydroJector-EM, which can replicate various time–pressure curves preloaded into the computer program before injection. The assessment of the reproducibility and safety of gene delivery system in vitro and in vivo demonstrated the precise replication of intravascular time–pressure curves and the reproducibility of gene delivery efficiency. The highest level of luciferase expression (272 pg luciferase per mg of proteins) was achieved safely using the time–pressure curve, which reaches 30 mm Hg in 10 s among various curves tested. Using this curve, the sustained expression of a therapeutic level of human factor IX protein (>500 ng ml−1) was maintained for 2 months after the HGD of the pBS-HCRHP-FIXIA plasmid. Other than a transient increase in liver enzymes that recovered in a few days, no adverse events were seen in rats. These results confirm the effectiveness of the HydroJector-EM for reproducible gene delivery and demonstrate that long-term therapeutic gene expression can be achieved by automatic computer-controlled hydrodynamic injection that can be performed by anyone.

Localization of exogenous DNA to mitochondria in skeletal muscle following hydrodynamic limb vein injection.

Mitochondrial genetic disorders are a major cause of mitochondrial diseases. It is therefore likely that mitochondrial gene therapy will be useful for the treatment of such diseases. Here, we report on the possibility of mitochondrial gene delivery in skeletal muscle using hydrodynamic limb vein (HLV) injection. The HLV injection procedure, a useful method for transgene expression in skeletal muscle, involves the rapid injection of a large volume of naked plasmid DNA (pDNA) into the distal vein of a limb. We hypothesized that the technique could be used to deliver pDNA not only to nuclei but also to mitochondria, since cytosolic pDNA that is internalized by the method may be able to overcome mitochondrial membrane. We determined if pDNA could be delivered to myofibrillar mitochondria by HLV injection by PCR analysis. Mitochondrial toxicity assays showed that the HLV injection had no influence on mitochondrial function. These findings indicate that HLV injection promises to be a useful technique for in vivo mitochondrial gene delivery.

Effective Prevention of Liver Fibrosis by Liver-targeted Hydrodynamic Gene Delivery of Matrix Metalloproteinase-13 in a Rat Liver Fibrosis Model

Liver fibrosis is the final stage of liver diseases that lead to liver failure and cancer. While various diagnostic methods, including the use of serum marker, have been established, no standard therapy has been developed. The objective of this study was to assess the approach of overexpressing matrix metalloproteinase-13 gene (MMP13) in rat liver to prevent liver fibrosis progression. A rat liver fibrosis model was established by ligating the bile duct, followed by liver-targeted hydrodynamic gene delivery of a MMP13 expression vector, containing a CAG promoter-MMP13-IRES-tdTomato-polyA cassette. After 14 days, the serum level of MMP13 peaked at 71.7 pg/ml in MMP13-treated group, whereas the nontreated group only showed a level of ~5 pg/ml (P < 0.001). These levels were sustained for the next 60 days. The statistically lower level of the hyaluronic acids in treated group versus the nontreated group (P < 0.05) reveals the therapeutic effect of MMP13 overexpression. Quantitative analysis of tissue stained with sirius red showed a statistically larger volume of fibrotic tissue in the nontreated group compared to that of MMP13-treated rats (P < 0.05). These results suggest that the liver-targeted hydrodynamic delivery of MMP13 gene could be effective in the prevention of liver fibrosis.

Liver-targeted hydrodynamic gene therapy: Recent advances in the technique

One of the major research focuses in the field of gene therapy is the development of clinically applicable, safe, and effective gene-delivery methods. Since the first case of human gene therapy was performed in 1990, a number of gene-delivery methods have been developed, evaluated for efficacy and safety, and modified for human application. To date, viral-vector-mediated deliveries have shown effective therapeutic results. However, the risk of lethal immune response and carcinogenesis have been reported, and it is still controversial to be applied as a standard therapeutic option. On the other hand, delivery methods for nonviral vector systems have been developed, extensively studied, and utilized in in vivo gene-transfer studies. Compared to viral-vector mediated gene transfer, nonviral systems have less risk of biological reactions. However, the lower gene-transfer efficiency was a critical hurdle for applying them to human gene therapy. Among a number of nonviral vector systems, our studies focus on hydrodynamic gene delivery to utilize physical force to deliver naked DNA into the cells in the living animals. This method achieves a high gene-transfer level by DNA solution injections into the tail vein of rodents, especially in the liver. With the development of genome editing methods, in vivo gene-transfer therapy using this method is currently the focus in this research field. This review explains the method principle, efficiency, safety, and procedural modifications to achieve a high level of reproducibility in large-animal models.

Active treatments are a rational approach for hepatocellular carcinoma in elderly patients

AIM To determine whether an active intervention is beneficial for the survival of elderly patients with hepatocellular carcinoma (HCC). METHODS The survival of 740 patients who received various treatments for HCC between 1983 and 2011 was compared among different age groups using Cox regression analysis. Therapeutic options were principally selected according to the clinical practice guidelines for HCC from the Japanese Society of Hepatology. The treatment most likely to achieve regional control capability was chosen, as far as possible, in the following order: resection, radiofrequency ablation, percutaneous ethanol injection, transcatheter arterial chemoembolization, transarterial oily chemoembolization, hepatic arterial infusion chemotherapy, systemic chemotherapy including molecular targeting, or best supportive care. Each treatment was used alone, or in combination, with a clinical goal of striking the best balance between functional hepatic reserve and the volume of the targeted area, irrespective of their age. The percent survival to life expectancy was calculated based on a Japanese national population survey. RESULTS The median ages of the subjects during each 5-year period from 1986 were 61, 64, 67, 68 and 71 years and increased significantly with time (P < 0.0001). The Child-Pugh score was comparable among younger (59 years of age or younger), middle-aged (60-79 years of age), and older (80 years of age or older) groups (P = 0.34), whereas the tumor-node-metastasis stage tended to be more advanced in the younger group (P = 0.060). Advanced disease was significantly more frequent in the younger group compared with the middle-aged group (P = 0.010), whereas there was no difference between the middle-aged and elderly groups (P = 0.75). The median survival times were 2593, 2011, 1643, 1278 and 1195 d for 49 years of age or younger, 50-59 years of age, 60-69 years of age, 70-79 years of age, or 80 years of age or older age groups, respectively, whereas the median percent survival to life expectancy were 13.9%, 21.9%, 24.7%, 25.7% and 37.6% for each group, respectively. The impact of age on actual survival time was significant (P = 0.020) with a hazard ratio of 1.021, suggesting that a 10-year-older patient has a 1.23-fold higher risk for death, and the overall survival was the worst in the oldest group. On the other hand, when the survival benefit was evaluated on the basis of percent survival to life expectancy, age was again found to be a significant explanatory factor (P = 0.022); however, the oldest group showed the best survival among the five different age groups. The youngest group revealed the worst outcomes in this analysis, and the hazard ratio of the oldest against the youngest was 0.35 for death. The survival trends did not differ substantially between the survival time and percent survival to life expectancy, when survival was compared overall or among various therapeutic interventions. CONCLUSION These results suggest that a therapeutic approach for HCC should not be restricted due to patient age.

Hemodynamics of a hydrodynamic injection

The hemodynamics during a hydrodynamic injection were evaluated using cone beam computed tomography (CBCT) and fluoroscopic imaging. The impacts of hydrodynamic (5 seconds) and slow (60 seconds) injections into the tail veins of mice were compared using 9% body weight of a phase-contrast medium. Hydrodynamically injected solution traveled to the heart and drew back to the hepatic veins (HV), which led to liver expansion and a trace amount of spillover into the portal vein (PV). The liver volumes peaked at 165.6 ± 13.3% and 165.5 ± 11.9% of the original liver volumes in the hydrodynamic and slow injections, respectively. Judging by the intensity of the CBCT images at the PV, HV, right atrium, liver parenchyma (LP), and the inferior vena cava (IVC) distal to the HV conjunction, the slow injection resulted in the higher intensity at PV than at LP. In contrast, a significantly higher intensity was observed in LP after hydrodynamic injection in comparison with that of PV, suggesting that the liver took up the iodine from the blood flow. These results suggest that the enlargement speed of the liver, rather than the expanded volume, primarily determines the efficiency of hydrodynamic delivery to the liver.

Site-Specific Impact of a Regional Hydrodynamic Injection: Computed Tomography Study during Hydrodynamic Injection Targeting the Swine Liver

A hemodynamic study of hydrodynamic gene delivery (HGD) from the tail vein in rodents has inspired a mechanism and an approach to further improve the efficacy of this procedure. However, there is no report on the hemodynamics of a regional HGD, which is an inevitable approach in large animals. Here, we report the hemodynamics of a regional hydrodynamic injection in detail based on 3D volume data and the dynamism of tissue intensity over time by using computed tomography (CT) both during and after a regional hydrodynamic injection that targeted the liver of a pig weighing 15.6 kg. Contrast medium (CM) was injected at a steady speed of 20 mL/s for 7.5 s under the temporal balloon occlusion of the hepatic vein (HV). A retrograde flow formed a wedge-shaped strong enhancement area downstream of the corresponding HV within 2.5 s, which was followed by drainage into another HV beginning from the target area and the portal vein (PV) toward a non-target area of the liver. After the injection, the CM was readily eliminated from the PV outside the target area. These data suggest that an interventional radiology approach is effective in limiting the hydrodynamic impacts in large animals at a target area and that the burden overflowing into the PV is limited. A further investigation that simultaneously evaluates gene delivery efficiency and hemodynamics using CT is needed to establish feasible parameters for a regional HGD in large animals.

Factors predicting aggressiveness of non-hypervascular hepatic nodules detected on hepatobiliary phase of gadolinium ethoxybenzyl diethylene-triamine-pentaacetic-acid magnetic resonance imaging

AIM To establish a prognostic formula that distinguishes non-hypervascular hepatic nodules (NHNs) with higher aggressiveness from less hazardous one. METHODS Seventy-three NHNs were detected in gadolinium ethoxybenzyl diethylene-triamine-pentaacetic-acid magnetic resonance imaging (Gd-EOB-DTPA-MRI) study and confirmed to change 2 mm or more in size and/or to gain hypervascularity. All images were interpreted independently by an experienced, board-certified abdominal radiologist and hepatologist; both knew that the patients were at risk for hepatocellular carcinoma development but were blinded to the clinical information. A formula predicting NHN destiny was developed using a generalized estimating equation model with thirteen explanatory variables: age, gender, background liver diseases, Child-Pugh class, NHN diameter, T1-weighted imaging/T2-weighted imaging detectability, fat deposition, lower signal intensity in arterial phase, lower signal intensity in equilibrium phase, α-fetoprotein, des-γ-carboxy prothrombin, α-fetoprotein-L3, and coexistence of classical hepatocellular carcinoma. The accuracy of the formula was validated in bootstrap samples that were created by resampling of 1000 iterations. RESULTS During a median follow-up period of 504 d, 73 NHNs with a median diameter of 9 mm (interquartile range: 8-12 mm) grew or shrank by 68.5% (fifty nodules) or 20.5% (fifteen nodules), respectively, whereas hypervascularity developed in 38.4% (twenty eight nodules). In the fifteen shrank nodules, twelve nodules disappeared, while 11.0% (eight nodules) were stable in size but acquired vascularity. A generalized estimating equation analysis selected five explanatories from the thirteen variables as significant factors to predict NHN progression. The estimated regression coefficients were 0.36 for age, 6.51 for lower signal intensity in arterial phase, 8.70 or 6.03 for positivity of hepatitis B virus or hepatitis C virus, 9.37 for des-γ-carboxy prothrombin, and -4.05 for fat deposition. A formula incorporating the five coefficients revealed sensitivity, specificity, and accuracy of 88.0%, 86.7%, and 87.7% in the formulating cohort, whereas these of 87.2% ± 5.7%, 83.8% ± 13.6%, and 87.3% ± 4.5% in the bootstrap samples. CONCLUSION These data suggest that the formula helps Gd-EOB-DTPA-MRI detect a trend toward hepatocyte transformation by predicting NHN destiny.

Effects of Fibrotic Tissue on Liver-targeted Hydrodynamic Gene Delivery

Hydrodynamic gene delivery is a common method for gene transfer to the liver of small animals, and its clinical applicability in large animals has been demonstrated. Previous studies focused on functional analyses of therapeutic genes in animals with normal livers and little, however, is known regarding its effectiveness and safety in animals with liver fibrosis. Therefore, this study aimed to examine the effects of liver fibrosis on hydrodynamic gene delivery efficiency using a rat liver fibrosis model. We demonstrated for the first time, using pCMV-Luc plasmid, that this procedure is safe and that the amount of fibrotic tissue in the liver decreases gene delivery efficiency, resulting in decrease in luciferase activity depending on the volume of fibrotic tissue in the liver and the number of hepatocytes that are immunohistochemically stained positive for transgene product. We further demonstrate that antifibrotic gene therapy with matrix metalloproteinase-13 gene reduces liver fibrosis and improves efficiency of hydrodynamic gene delivery. These results demonstrate the negative effects of fibrotic tissue on hydrodynamic gene delivery and its recovery by appropriate antifibrotic therapy.Hydrodynamic gene delivery is a common method for gene transfer to the liver of small animals, and its clinical applicability in large animals has been demonstrated. Previous studies focused on functional analyses of therapeutic genes in animals with normal livers and little, however, is known regarding its effectiveness and safety in animals with liver fibrosis. Therefore, this study aimed to examine the effects of liver fibrosis on hydrodynamic gene delivery efficiency using a rat liver fibrosis model. We demonstrated for the first time, using pCMV-Luc plasmid, that this procedure is safe and that the amount of fibrotic tissue in the liver decreases gene delivery efficiency, resulting in decrease in luciferase activity depending on the volume of fibrotic tissue in the liver and the number of hepatocytes that are immunohistochemically stained positive for transgene product. We further demonstrate that antifibrotic gene therapy with matrix metalloproteinase-13 gene reduces liver fibrosis and improves efficiency of hydrodynamic gene delivery. These results demonstrate the negative effects of fibrotic tissue on hydrodynamic gene delivery and its recovery by appropriate antifibrotic therapy.