A Surgical Model for Isolating the Pig Liver in vivo for Gene Therapy

Abstract

Several studies report results that suggest the need of vascularization blocking for efficient gene transfer to the liver, especially in nonviral gene therapy. In this study, we describe a surgical strategy for in vivo isolation of the pig liver, resulting in a vascular watertight organ that allows the evaluation of several gene injection conditions. The hepatic artery and portal, suprahepatic and infrahepatic cava veins were dissected. Then, liver vascularization was excluded for 5-7 min. In that time, we first injected 200 ml saline solution containing the p3c-eGFP plasmid (20 µg/ml) simultaneously through two different catheters placed in the portal and cava veins, respectively. Vital constants were monitored during the surgery to assess the safety of the procedure. Basal systolic/diastolic blood pressures were 92.8/63.2 mm Hg and dropped to 40.7/31.3 mm Hg at the end of vascular exclusion; the mean basal heart rate was 58 bpm, reaching 95 bpm when the blood pressure was low. Oxygen saturation was maintained above 98% during the intervention, and no relevant changes were observed in the ECG tracing. Peak plasma AST (aspartate aminotransferase) and ALT (alanine aminotransferase) levels were observed after 24 h (151 and 57 IU, respectively). These values were higher, but not relevant, in 60 ml/s injection than in 20 ml/s injection. Efficiency of gene transfer was studied with simultaneous (cava and portal veins) injection of eGFP gene at flow rates of 20 and 60 ml/s. Liver tissue samples were collected 24 h after injection and qPCR was carried out on each lobe sample. The results confirmed the efficiency of the procedure. Gene delivery differed between 20 ml/s (9.9-31.0 eGFP DNA copies/100 pg of total DNA) and 60 ml/s injections (0.6-1.1 eGFP DNA copies/100 pg of total DNA). Gene transcription showed no significant differences between 20 ml/s (15,701.8-21,475.8 eGFP RNA copies/100 ng of total RNA) and 60 ml/s (12,014-36,371 eGFP RNA copies/100 ng of total RNA). The procedure is not harmful for animals and it offers a wide range of gene delivery options because it allows different perfusion ways (anterograde and retrograde) and different flow rates to determine the optimal conditions of gene transfer. This strategy permits the use of cell therapy and viral or non-viral liver gene therapy, especially appropriated to a wide variety of inherited or acquired diseases because of the liver's ability to produce and deliver proteins to the bloodstream.

Human AAT gene transfer to pig liver improved by using a perfusion isolated organ endovascular procedure

“…In this context, high flow rates (40-100 ml/s) resulted in low transfection efficiency, whereas, in our experience [28], lower injection flow rates (10-20 ml/s) resulted in transcription indexes equivalent to genes with low-intermediate expression activity. These findings agree with the efficacy of gene transfer to watertight human liver segments [20], employing mild injection flow rates (10 ml/s).…”

“…After achieving efficient gene transcription following gene transfer in the surgically isolated pig liver in vivo [28], we aimed to establish a minimally invasive procedure for liver isolation by endovascular catheterization. Endovascular procedures offer advantages in terms of their potential clinical applications.…”

Safety Assessment of Liver-Targeted Hydrodynamic Gene Delivery in Dogs

“…While effective and safe in small animals, the major challenge is in applying this simple procedure in human gene therapy. For this purpose, a few groups have reported some success in rabbits and small pigs (<20 kg) [8], [9], [21]–[26]. We have reported catheter-based, regional hydrodynamic gene delivery to transfer reporter plasmids to a specific region of a target organ by inserting the catheter into a selected blood vessel [8], [9], [11].…”

Liver Gene Therapy: Employing Surgery and Radiology for Translational Research

“…As previously reported, the complete liver vascularization of the pig can be occluded up to 20 min without neither hepatic injury nor systemic damage [52]. Considering this fact, Carreño et al [50] described in pigs a surgical procedure to completely exclude liver vascularization 'in vivo' and perform hydrodynamic gene delivery, targeting the entire organ. A complete midline laparotomy was carried out, exposing all the abdominal organs.…”

“…Aiming to achieve localized high pressure without affecting the systemic circulation, they proposed individualizing the lobe by employing catheters with balloon and ligation. Although most of the authors suggested blood pressure to be the most important feature of hydrodynamic injection for efficient gene transfer, others have pointed other characteristics such as impulse [49] and flow rate [50,51] to be relevant. However, nearly all authors agree to the need for isolating target areas or the entire liver to improve the procedure efficiency.…”