Greta J. Sawyer

Hydrodynamic gene delivery to the pig liver via an isolated segment of the inferior vena cava.

Hydrodynamic gene delivery is an attractive option for non-viral liver gene therapy, but requires evaluation of efficacy, safety and clinically applicable techniques in large animal models. We have evaluated retrograde delivery of DNA to the whole liver via the isolated segment of inferior vena cava (IVC) draining the hepatic veins. Pigs (18–20 kg weight) were given the pGL3 plasmid via two programmable syringe pumps in parallel. Volumes corresponding to 2% of body weight (360–400 ml) were delivered at 100 ml s−1 via a Y connector. The IVC segment pressure, portal venous pressure, arterial pressure, electrocardiogram (ECG) and pulse were monitored. Concurrent studies were performed in rats for interspecies comparisons. The hydrodynamic procedure generated intrahepatic vascular pressures of 101–126 mm Hg, which is ∼4 times higher than in rodents, but levels of gene delivery were ∼200-fold lower. Suprahepatic IVC clamping caused a fall in arterial pressure, with the development of ECG signs of myocardial ischaemia, but these abnormalities resolved rapidly. The IVC segment approach is a clinically acceptable approach to liver gene therapy. However, it is less effective in pigs than in rodents, possibly because of larger liver size or a less compliant connective tissue framework.

Comparison of adenovirus gene transfer to vascular endothelial cells in cell culture, organ culture, and in vivo.

A replication-defective adenovirus 5 vector carrying the beta-galactosidase reporter gene was tested for its efficiency for gene delivery to vascular endothelial cells in various situations. Both porcine and human primary vascular endothelial cell cultures were very efficiently infected (>90%) at adenovirus concentrations of 10(10) pfu/ml or higher. Cultured rat fibroblasts and keratinocytes were even more readily infected, with >90% infection with adenovirus titers of 10(8) pfu/ml or higher. However, nondividing vascular endothelium in situ was very poorly transduced. Pieces of aorta from adult pigs, sheep, rabbit and rat, and pieces of human umbilical artery and vein were studied in organ culture. These showed only occasional positive vascular endothelial cells when exposed to the adenovirus vector at concentrations up to 5x10(11) pfu/ml. Kidney perfusion studies in rats and pigs gave similar results. The only exception to the above findings was in very young (3-4 day old) piglets, which showed excellent (>90%) infection of vascular endothelium with the adenovirus vector at titers of 10(10) pfu/ml. Our data suggest that adenovirus vectors will not be of value for gene delivery to uninjured vascular endothelium in situ, and are therefore unsuited for ex vivo genetic manipulation of vascular endothelium in organs for transplantation.

Regional hydrodynamic gene delivery to the rat liver with physiological volumes of DNA solution.

The major barrier to the clinical application of hydrodynamic gene delivery to the liver is the large volume of fluid required using standard protocols. Regional hydrodynamic gene delivery via branches of the portal vein has not previously been reported, and we have evaluated this approach in a rat model.

A detailed analysis of the potential of water-soluble classical class I MHC molecules for the suppression of kidney allograft rejection and in vitro cytotoxic T cell responses

Water-soluble classical (RT1-A) class I MHC molecules were purified from aqueous extracts of DA strain liver. Following monoclonal antibody affinity, lentil lectin affinity, and gel filtration chromatography, 600 micrograms of soluble RT1-A class I molecules with antigen activity equivalent to 1.3 x 10(11) nucleated DA spleen cells (greater than 500 DA spleens) was obtained. Both PVG and LEW strain recipients of DA kidney allografts were pretreated with intravenous injections of the DA soluble class I molecules, in doses with antigen activity equivalent to 10(8) nucleated DA spleen cells. Three protocols of pretreatment were used: twice-weekly injections for 4-5 weeks, with grafting 3 or 4 days after the last injection; a single injection 7 days pregraft; or a single injection 1 day pregraft. The PVG and LEW rats received the soluble class I pretreatment either alone or in combination with suboptimal doses (2 mg/kg/day) of cyclosporine after grafting, making a total of 12 experimental groups treated with soluble class I antigen. In no case did treatment with soluble class I antigen elicit an antibody response in prospective graft recipients; influence kidney graft survival in any way; or enhance or suppress the antibody response to the kidney graft. The soluble DA class I MHC molecules were tested in vitro for their effect on the generation and effector function of allospecific PVG and LEW anti DA RT1-A class I cytotoxic T cells and TNP specific, self RT1-Aa restricted cytotoxic T cells. Concentrations up to 5 micrograms/ml (10(-7) M), equivalent to 10(9) nucleated DA spleen cells/ml, were without any effect. We conclude that monomeric forms of water-soluble classical class I molecules are poor immunogens--and, at doses conventionally used for active enhancement, do not influence cytotoxic T cell responses and have little potential for donor-specific immunosuppression.

In Vivo Gene Delivery via Portal Vein and Bile Duct to Individual Lobes of the Rat Liver Using a Polylysine-Based Nonviral DNA Vector in Combination with Chloroquine

The objective of this study was to evaluate a bifunctional synthetic peptide as a DNA vector for regional gene delivery to the rat liver by the portal vein and bile duct routes. The 31-amino-acid peptide (polylysine-molossin) comprises an amino-terminal chain of 16 lysines for electrostatic binding of DNA, and the 15 amino acid integrin-binding domain of the venom of the American pit viper, Crotalus molossus molossus. Initial in vitro evaluation demonstrated that polylysine-molossin/DNA complexes were much smaller (approximately 50-100nm versus 500-1300nm), more positively charged, and more stable in isotonic dextrose in comparisons with salt-containing solutions. However, polylysine-molossin/DNA complexes in any solution other than complete culture medium were ineffective for gene delivery in vitro. Vector localization studies demonstrated that both the portal vein and bile duct routes provided excellent access of polylysine-molossin/DNA complexes to the liver. However, complexes delivered by the portal vein were rapidly lost (<15 min) following re-establishment of the portal circulation, whereas complexes delivered by the bile duct persisted much longer. Polylysine-molossin/DNA complexes in various isotonic solutions were delivered to the right lateral lobes either by perfusion through a branch of the portal vein or by infusion into appropriate branches of the bile duct. Two or three hours before gene delivery, rats were given a single injection of chloroquine. We report that the polylysine-molossin vector is much more effective (>10-fold) when delivered by the bile duct route with all isotonic solutions evaluated, and that polylysine-molossin/DNA complexes in isotonic dextrose are much more effective (>10-fold) than complexes in salt-containing solutions.

Hydrodynamic Gene Delivery to the Liver: Theoretical and Practical Issues for Clinical Application

Hydrodynamic gene delivery to the liver has potential as a safe and effective approach for clinical liver gene therapy. However, the simplicity of the technique in rodents - an intravenous injection - belies the theoretical and practical complexity for clinical application. A key issue is that outflow obstruction of the DNA solution from the liver is a critical factor for raising intrahepatic vascular pressure, which in turn provides the force to swell the liver and effect gene delivery. For conventional hydrodynamic gene delivery via tail vein injection, this outflow obstruction is provided naturally by the vascular resistance of the gut, spleen and pancreas. For regional hydrodynamic gene delivery to the liver, outflow obstruction to create a closed system requires surgical intervention, making it unlikely that minimally invasive techniques will be possible in the clinic. Intrinsic factors, in particular compliance (elasticity) of the liver are likely to be crucial in determining the degree of swelling for a given level of intrahepatic vascular pressure. Liver compliance is likely to be the major reason for the low level of hydrodynamic gene delivery in the pig model, and will influence the effectiveness of the approach in man, both in general and in different disease states.

Pancreatic duodenal homeobox 1 expression is insufficient to transdifferentiate liver cells into insulin-secreting cells

Objective: To compare the preoperative computed tomography (CT) findings in patients with potentially operable pancreatic malignancy with findings at surgery. Methods: In a retrospective analysis, 140 consecutive patients with carcinoma of the pancreatic head were studied. All were imaged using a standardized multidetector CT (MDCT) protocol. Patients with disease that was clearly inoperable were excluded. The remaining patients had their CT studies double-reported using a standard method. Images were scored for vascular involvement, tumor size, nodal disease, pancreatic duct diameter, and size of the gastrocolic trunk. Preoperative staging was compared with findings at surgery. Results: One hundred forty patients presented with pancreatic head tumors. One hundred were not suitable for surgery. Forty patients were considered for curative surgery. For assessing preoperative operability, MDCT has an accuracy, sensitivity, specificity, positive predictive value, and negative predictive value of 72.7, 81.8, 68.2, 56, and 88.2%, respectively. Subjects with inoperable tumors tended to have larger tumors and more dilated pancreatic ducts (P = 0.04). Conclusions: There remains a group of patients with small pancreatic tumors that show early local dissemination, undetectable with high-resolution anatomical imaging.

In vitro investigation of factors important for the delivery of an integrin-targeted nonviral DNA vector in organ transplantation.

BACKGROUND Polylysine-molossin is a 31 amino acid synthetic peptide that has previously been demonstrated to function as a DNA vector in vitro for cell lines and for the cornea. It incorporates the 15 amino acid integrin-binding domain of the venom of the American pit viper, Crotalus molossus molossus as the targeting moiety and a chain of 16 lysines as the DNA-binding moiety. The objective of this study was to evaluate several parameters of importance for in vivo applications. METHODS Binding and tissue distribution of the vector/DNA complexes were followed by a monoclonal antibody to the vector, or by the use of fluorescein-labeled DNA. Standard in vitro transfections were used to monitor effective gene transfer. RESULTS (1) Optimal DNA/vector concentration. Saturation of vector/DNA binding sites on the ECV304 cell line occurred at 6 microg/ml of DNA. The concentration of vector/DNA complexes required for optimal gene transfection was found to be 2-8 microg/ml of DNA, corresponding to the concentration needed for saturation binding. (2) Optimal target cell exposure time. Vector/ DNA complexes saturated target cell binding sites within 5 min of incubation. However, lengthy exposure times (>2-3 hr) to the transfection medium were essential for substantial gene transfer. This was a consequence of two complementary factors. First, it was important that target cells be exposed to vector/DNA complexes for approximately 1 hr at 37 degrees C. Saturation of target sites at 4 degrees C and then removal of the transfection medium was much less effective. Second, exposure to chloroquine for 8-10 hr after uptake of vector/DNA complexes was essential for optimal gene transfer. (3) Inhibitory effects of serum. Exposure of complexes to even 1% serum before transfection, markedly inhibited gene transfer. However, target cells previously saturated with vector/DNA complexes and then exposed to 10% serum showed substantial gene transfer. (4) Extravasation and binding stability in vivo. Cold ex vivo perfusion of rat hearts with vector/DNA complexes demonstrated that little, if any, complex moved out of the vascular system. After transplantation of the heart, most of the complex bound to the vasculature was lost within 30 min of reestablishing the blood circulation. CONCLUSIONS Careful attention to several parameters of little importance in vitro need to be paid for optimal in vivo application of DNA vector systems.

Cardiovascular function following acute volume overload for hydrodynamic gene delivery to the liver

Hydrodynamic gene delivery to the liver is a valuable experimental tool and an attractive option for nonviral gene therapy of liver disease. However, little attention has been paid to the major obstacle to clinical application: acute volume overload of the cardiovascular system. We delivered volumes of DNA solution (pGL3 plasmid) corresponding to 1, 2, 4, 6 and 8% of the body weight at 100 ml/min to the inferior vena cava (IVC) of DA strain rats. Central venous pressure (CVP), arterial pressure, pulse and electrocardiogram (ECG) were continuously recorded for subsequent analysis. Each volume produced a characteristic response, but all (including the 1% volume) caused severe falls in blood pressure and pulse within 1–2 s of the infusion, with ectopic beats and widening of the QRS complex in the ECG. The response to volumes of 4% and higher suggested that the liver acted as a volume sink, mitigating the immediate effects of volume overload. The 6 and 8% volumes caused profound and protracted falls in blood pressure and pulse, with a multitude of severe electrical abnormalities in the heart, including electromechanical dissociation. Vagal blockade with atropine, and the use of Ringers solution to prevent electrolyte disturbances, did not ameliorate this picture.

Suppression of kidney allograft rejection across full MHC barriers by recipient-specific antibodies to class II MHC antigens.

The aim of these studies was to see if recipient-specific antibodies to class II MHC antigens might be effective in suppressing kidney graft rejection in rats. For these experiments, the polymorphic BMAC-4 mouse IgG1 monoclonal antibody to RT1-D class II MHC antigens was raised. This antibody reacts with the DA, LEW, PVG, and SHR strains, but not the BN or WAG strains, and is therefore recipient-specific in the WAG to PVG combination. Initial in vivo titrations demonstrated that 1 ml doses of the BMAC-4 and also of the MRC OX6 (monomorphic mouse IgG1 anti-RT1-B class II) antibody resulted in the maintenance of free antibody levels in blood for > 24hr. Treatment of PVG recipients of WAG kidney allografts with the BMAC-4 antibody, but not the MRC OX6 antibody, resulted in greatly prolonged graft survival. To examine possible mechanisms, several experiments were performed. After intravenous injection, the antibody was found to have ready access to the connective tissues of nonlymphoid organs, to the red and white pulp of the spleen, and to the medulla of lymph nodes. However, there was poor early access to the cortex and paracortex of lymph nodes. Both MRC OX6 and BMAC-4 could completely suppress PVG anti-WAG and WAG anti-PVG mixed lymphocyte culture reactions. Both antibodies were also equally effective for opsonisation of class II-positive cells from the blood circulation. However, only the recipient-specific, anti RT1-D BMAC-4 antibody suppressed graft rejection. Thus, while the BMAC-4 antibody is likely to have had a variety of different effects on RT1-D positive recipient cells, the locus specificity of the immunosuppression is consistent with an important component of those effects being the blocking of presentation of WAG donor alloantigens by PVG RT1-D class II antigens on PVG antigen-presenting cells.