DNA delivery to ‘ex vivo’ human liver segments

Abstract

Hydrodynamic injection is an efficient procedure for liver gene therapy in rodents but with limited efficacy in large animals, using an 'in vivo' adapted regional hydrodynamic gene delivery system. We study the ability of this procedure to mediate gene delivery in human liver segments obtained by surgical resection. Watertight liver segments were retrogradely injected from hepatic vein with a saline solution containing a plasmid bearing the enhanced green fluorescent protein (eGFP) gene, under different conditions of flow rate (1, 10 and 20 ml s -1 ) and final perfused volume. Samples were cultured for 1 to 2 days and used for microscopy and molecular analysis of gene expression. The fluorescent and immunohistochemistry studies indicated that in segments injected at X10 ml s -1 , good and wide gene expression was present in the liver sections and the molecular analysis reinforced the histological observation in a quantitative manner (index of apparent gene delivery: 10 2 -10 4 eGFP DNA copy per 100 pg of total DNA; transcription index: 10 5 -2Â10 6 eGFP RNA copy per 100 ng of total RNA). In addition, injected gold nanoparticles (15 nm diameter) suggested that DNA delivery to hepatocytes must involve a facilitated permeation process without membrane disruption. In summary, we show that retrograde venous injection of watertight human liver segment is an anadromous procedure that results in wide liver gene delivery and good gene expression. However, additional studies will be necessary to clarify the influence of the prolonged ischemia injury to hepatocytes in our model.

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). We decided to inject the DNA solution retrovenously since it has been demonstrated in other organs, such as the heart, that the procedure achieving the highest efficiency is mediated by retrovenous gene transfer.…”

“…Despite the great effort and the different strategies developed to date, all of them have resulted in protein expression levels far lower than those observed in small animals. However, the perfusion of individualized and watertight human liver segments for retrograde gene flow transfer has also obtained great efficiency [20], suggesting that complete liver vascular arrestment could be necessary to achieve expression of the transferred gene.…”

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

“…Protein expression occurs mainly in the perfused area. Our group [18] followed this line, employing watertight human liver segments. The latter were obtained from surgical resection, and consequently resulted in vascular watertight tissue.…”

Hydrodynamic Delivery

“…Studies focused on the identification of factors in large animals that interfere with gene transfer and expression are in great need before we can develop a new generation of injection devices for clinical evaluation of hydrodynamic gene therapy. The information generated from swine (Alino, Herrero, Noguera, Dasi, & Sanchez, 2007;Fabre et al, 2008;Yoshino, Hashizume, & Kobayashi, 2006;Kamimura et al, 2009;Kamimura et al, 2010), dogs (Kamimura et al, unpublished results), and isolated human liver (Herrero et al, 2012) appears critical for design of another set of experimentation that may pave the way for clinical application of HD.…”