Development of alternative gene transfer techniques for ex vivo and in vivo gene therapy in a canine model

Abstract

Introduction: Gene therapy have recently attracted much attention as a curative therapeutic option for inherited single gene disorders such as hemophilia. Hemophilia is a hereditary bleeding disorder caused by the deficiency of clotting activity of factor VIII (FVIII) or factor IX (FIX), and gene therapy for hemophilia using viral vector have been vigorously investigated worldwide. Toward further advancement of gene therapy for hemophilia, we have previously developed and validated the efficacy of novel two types of gene transfer technologies using a mouse model of hemophilia A. Here we investigated the efficacy and safety of the technologies in canine model. Especially, validations of technical procedures of the gene transfers for dogs were focused.

Methods: Green fluorescence protein (GFP) gene were transduced into normal beagle dogs by ex vivo and in vivo gene transfer techniques. For ex vivo gene transfer, blood outgrowth endothelial cells (BOECs) derived from peripheral blood of normal dogs were transduced with GFP gene using lentivirus vector, propagated, fabricated as cell sheets, then implanted onto the omentum of the same dogs. For in vivo gene transfer, normal dogs were subjected to GFP gene transduction with non-viral piggyBac vector by liver-targeted hydrodynamic injections.

Results: No major adverse events were observed during the gene transfers in both gene transfer systems. As for ex vivo gene transfer, histological findings from the omental biopsy performed 4 weeks after implantation revealed the tube formation by implanted GFP-positive BOECs in the sub-adipose tissue layer without any inflammatory findings, and the detected GFP signals were maintained over 6 months. Regarding in vivo gene transfer, analyses of liver biopsy samples revealed more than 90% of liver cells were positive for GFP signals in the injected liver lobes 1 week after gene transfers, then the signals gradually declined overtime.

Conclusions: Two types of gene transfer techniques were successfully applied to a canine model, and the transduced gene expressions persisted for a long term. Toward clinical application for hemophilia patients, practical assessments of therapeutic efficacy of these techniques will need to be performed using a dog model of hemophilia and FVIII (or FIX) gene.

Keywords: BOEC, blood outgrowth endothelial cell; Cell sheet; Dog; FIX, factor IX; FVIII, factor VIII; GFP, green fluorescent protein; Gene therapy; Hemophilia; Hydrodynamic injection.

Fig. 1

Lentiviral vector and piggyBac transposon vector. Schematic diagram of lentiviral vector (A) and piggyBac transposon vector (B) expressing GFP under the control of the human EF1α promoter. IRES: internal ribosomal entry site.

Fig. 2

Lentiviral vector transduction of BOECs in vitro. Canine BOECs were transduced with Lenti-EF1α-GFP at various multiplicity of infections (MOIs), and the percentage of BOECs expressing GFP was calculated by flow cytometry at 3 days after transduction. At MOI of 20, 2, 1, and 0.5, 93.4%, 60.0%, 43.7%, and 27.4% of BOECs expressed GFP. The percentage of GFP-positive cells of non-transduced BOECs was 3.1%.

Fig. 3

Implantation of genetically-modified blood outgrowth endothelial cells (BOECs) sheets on the omentum of dogs. Two beagle dogs (dog 1 and 2) were implanted with autologous BOECs sheets which were transduced with GFP gene ex vivo, on their omentum. (A) The macroscopic views of the omentum of recipient dog 1 immediately after cell sheet implantation. For future biopsy, implanted areas were marked with nylon non-absorbable surgical suture (blue color) for orientation. (B) The macroscopic views of the omentum of dog 1 at 4 weeks after implantation (the timing of first biopsy). (C–E) Histological analysis from the ometum biopsy samples. H&E staining (x10) at 4 weeks (C), GFP fluorescence at 4 weeks (x80) (D), and GFP fluorescence at 20 weeks after implantation (x10) are shown. GFP view at 4 weeks was focused on the parts of omentum indicated by open square. The implanted BOECs could efficiently engrafted, partly differentiated into mature endothelial cells and formed new blood vessel structures within recipient omentum without reducing GFP gene expressions.

Fig. 4

Liver-targeted gene transfer by hydrodynamic injection on dogs. Two beagle dogs (Dog 3 and 4) were subjected to in vivo GFP gene transduction with piggyBac vector by liver-targeted hydrodynamic injection. The prepared plasmid mixture was divided into three (50 mL, each), and then injected into the 3 liver lobes, right lateral, right medial, and left lateral lobe, respectively, at the speed of 10 mL per second for 5 s. Macroscopic views of the liver of dog 3 before (A) and immediately after (B) three times of injections are shown. An obvious overexpansion of liver lobes was observed after hydrodynamic injections.

Fig. 5

Change of physiological parameters during the liver-targeted gene transfer by hydrodynamic injection on dogs. Heart rate (filled square), systolic blood pressure (filled circle), and diastolic blood pressure (open circle) of dog 3 and 4 were monitored during the hydrodynamic injection procedures. Filled arrows indicate the timing of hydrodynamic injections. The slight elevations of blood pressures accompanied with bradycardia were recorded immediately after hydrodynamic injections but returned to the baseline during the interval without any medical treatments.

Fig. 6

Electrocardiogram during the liver-targeted gene transfer by hydrodynamic injection on dogs. Electrocardiogram of dog 3 prior, during, 3 min, and 10 min after hydrodynamic injection to the liver lobe are shown. No changes in the dynamic ST segments and T waves were observed.

Fig. 7

Assessments of liver function by serum biochemistry during the liver-targeted gene transfer by hydrodynamic injection on dogs. Blood samples were collected from the cephalic veins of dog 3 and 4 before, and 4, 24, 168, 336 h after hydrodynamic injections. Serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were measured. Open squares and filled squares indicate dog 3 and 4, respectively. Transient increases of AST and ALT were observed within 24 h after hydrodynamic injection, especially in dog 4, but these levels were normalized within 168 h without any medical treatments.

Fig. 8

Histological findings from the liver biopsy of dogs after liver-targeted gene transfer by hydrodynamic injection. Gene delivery efficiency was assessed by detecting GFP expression of the liver biopsy samples collected at 1 and 20 weeks after the hydrodynamic injections. Representative photomicrographs of liver histology of dog 3 obtained 1 week after injections were shown. Hematoxylin and eosin (H&E) staining (x20) (A&C) and GFP expressions (x20) (B&D) of right medial liver lobe (injected lobe) (A&B) and right lateral liver lobe (non-injected lobe) (C&D) were shown. Approximately over 90% of liver cells in injected liver lobes were positive for GFP signals, whereas GFP signal were scarcely detected in non-injected lobes. HE staining demonstrated no significant histological findings such as the deformation of hepatocytes and the expansion of the sinusoids.