Novel Gene Therapy Strategy for Hemophilia a By Hydrodynamic Gene Delivery Combined with Non-Viral Piggybac Transposon Vector in Canine Model

Currently, hemophilia A patients are treated with plasma-derived or recombinant factor VIII (FVIII) concentrates. These treatments have substantially improved the management of bleedings in hemophilia and more recently long-acting recombinant FVIII concentrates are available in clinic, but hemophilia A patients still require the repeated intravenous injections frequently 2-3 times per week. Therefore, gene and cell-based therapy is an attractive strategy for the treatment of the disease because continuous expression of FVIII levels as low as 1-5 % of normal have been shown to ameliorate the bleeding phenotype as well as improve the quality of life. Although several attempts have been made for hemophilia A gene therapy, systemic delivery of viral vectors into liver cells in vivo, however, resulted in a transient and low-level expression of FVIII, because the large size of FVIII cDNA prohibited the efficient delivery of conventional viral vectors. Moreover, there remain concerns over the safety of these approaches such as adverse immunological reactions or virus-mediated cytotoxicity. Hydrodynamic gene delivery (HGD) is considered to be an alternative candidate to viral vectors. Previously, we tested the HGD in hemophilia A mouse model with the combination of non-viral piggyBac transposon vectors which can transfer the full-length FVIII transgene. As a result, we confirmed the sustained FVIII expression for over 300 days without any immune responses (Matsui H, et.al. PLos One 9(8) e104597, 2014).

While HGD system is effective and safe in small animal such as mouse (20-25g body weight), the major challenge is in applying this functioning procedure in the patients with hemophilia A. For this purpose, the current study focus on an assessment of the safety of liver-target HGD in dogs as the first step toward hydrodynamic gene therapy in clinic. Hemophilia A carrier dog was provided from Dr. Lillicrap group at Queen's University and we have already developed hemophilia A dog colony in our institution. We used both normal and hemophilia A dogs in this study. In brief, under general anesthesia using isoflurane, beagle dog (10-12Kg body weight) was placed on the table of fluoroscopy machine. A 18G peripheral catheter was directly inserted into the saphenous vein followed by insertion of a 0.038 inch guide wire, a long sheath (5Fr) and an injection balloon catheter. The balloon catheter was placed and inflated just below the branch of hepatic vein using the image-guided interventional radiology technique. Liver-targeted HGD was performed into 4 liver lobes, the right lateral, right medial, left medial, and left lateral lobe, separately using piggyBac transposon vector expressing either GFP or FVIII. An injection of saline containing plasmid DNA were performed at the speed of 10mL/second sequentially into 4 liver lobes through the hepatic veins with 20 minutes interval between each injections.Various physiological parameters including heart rate, blood pressure, oxygen saturation were continuously monitored and recorded before, during and after each injection. For the assessment of tissue damage, blood samples were collected at the various time points. For the assessment of GFP expression, the liver samples were collected under echo-guided liver biopsy both 5 and 120 days after HGD. Although, transient 5-10 fold increase in hepatobiliary enzymes, including AST, ALT, were observed, there is no significant impact on physiological parameters, such as systemic circulation, respiration and cardiac function.By histological examination of liver samples, approximately 70-80% of hepatocytes in all 4 lobes were positive with GFP both 5 and 120 days after HGD. These results suggest that HGD system combined with interventional radiology technique is capable for the large animal with safe, efficient and less invasive. Now, we are conducting FVIII gene transfer by HGD in canine hemophilia A model.