Technical requirements for effective regional hydrodynamic gene delivery to the left lateral lobe of the rat liver.

Hydrodynamic gene delivery to the liver is an attractive approach for clinical liver gene therapy, but critical aspects of technique remain uncertain. There has not been to date any report of high levels of hydrodynamic gene delivery to the liver, except in rodents. Regional hydrodynamic delivery to individual lobes/segments of the liver is being pursued in preclinical pig models, where reporter gene expression has been <1% of rodent levels, and in one clinical study, where there was no substantive evidence of gene expression. In none of these studies did surgical technique include outflow obstruction of the DNA solution. Here we report a novel technique for regional hydrodynamic gene delivery to the left lateral lobe of the rat liver. The technique gives high levels of gene delivery specific to the left lateral lobe with low volumes ( approximately 1.5 ml) of DNA solution, and permits an evaluation of hydrodynamic delivery in the presence and in the absence of outflow obstruction. We report that outflow obstruction is an absolute requirement for effective hydrodynamic gene delivery to individual lobes/segments of the liver, and therefore that minimally invasive techniques will not be possible in the clinic.

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 approximately 4 times higher than in rodents, but levels of gene delivery were approximately 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.

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 Ringer's solution to prevent electrolyte disturbances, did not ameliorate this picture.

A remarkable permeability of canalicular tight junctions might facilitate retrograde, non-viral gene delivery to the liver via the bile duct.

AIMS: To establish the extent of retrograde bile duct infusion at an ultrastructural level, as a preliminary step before evaluating the efficacy of gene delivery to the rat liver via a branch of the bile duct. METHODS: The extent of retrograde infusion into the biliary tree was established by light and electron microscopy, following infusion of 10 nm gold particles into the right lateral lobe. Canalicular permeability was further assessed by the infusion of a 67 kDa protein. For gene delivery, both naked DNA and a synthetic peptide vector system were evaluated. Because canalicular tight junction permeability can be compromised in damaged livers, both normal rats and rats recovering from the hepatotoxin D-galactosamine were studied. RESULTS: The gold particles penetrated the peripheral one third of the hepatic lobules and, surprisingly, reached the space of Disse in normal rats. Equally surprisingly, blood levels of a 67 kDa protein were identical after bile duct infusion and portal vein injection. Gene delivery with peptide/DNA complexes was much more effective in rats treated with D-galactosamine. However, gene delivery with naked DNA was equally effective in normal and damaged livers. Localisation of gene expression showed a scattering of positive hepatocytes restricted to the right lateral lobe. CONCLUSIONS: Retrograde infusion into the bile duct advances well into the hepatic lobule and reveals a remarkable permeability of the canalicular or cholangiole tight junctions in normal rats. It is an effective approach for delivering genes to a small population (approximately 1%) of hepatocytes.

A small, synthetic peptide for gene delivery via the serpin-enzyme complex receptor.

BACKGROUND: The serpin-enzyme complex receptor (SECR) has previously been successfully targeted for gene delivery using synthetic peptide ligands covalently linked in fluid phase to commercially available polylysine preparations (approximately 10-54kDa). The objective of the present study was to improve this approach by the use of small, bifunctional, and easily standardised synthetic peptides. METHODS: Two synthetic peptides designated polylysine antitrypsin 1 (PAT1) (K16 FNKPFVFLI) and PAT2 (K16 CSIPPEVKFNKPFVFLI) were evaluated for gene delivery to the HUH7 human hepatocyte cell line. The K16 moiety binds DNA electrostatically, while the FVFLM motif of human alpha1-antitrypsin targets the SECR. RESULTS: Both PAT1 and PAT2 bind to and condense DNA into small particles as shown by laser scattering techniques. However, only PAT2 is effective for gene delivery, presumably on account of the greater distance between the K16 chain and the FVFLM motif. Gene delivery by PAT2/DNA complexes is chloroquine-dependent, can be blocked completely by free ligand (CSIPPEVKFNKPFVFLI), and is highly efficient (e.g. approximately five-fold more effective than lipofectamine). At physiological salt concentrations, PAT2/DNA complexes formed at 4 microg/ml DNA are approximately 350 nm in diameter and highly effective for gene transfer, but at 100 microg/ml DNA the complexes are aggregated (diameter > 4 microm) and inactive. CONCLUSIONS: A small (33 amino acid), bifunctional, synthetic peptide represents a highly efficient and readily standardised DNA vector for the SECR. The effectiveness of this peptide depends on the distance of the K16 moiety from the targeting ligand. High salt concentrations are not required to form effective vector/DNA complexes.

The allogeneic response and tumor immunity.

The strong allogeneic response to donor MHC molecules in transplantation and the weak response to tumor antigens represent two important and divergent but potentially interactive immune responses. A patient's response to allogeneic MHC molecules might promote an effective T-cell response to self MHC-restricted tumor peptides and the possibilities for this are discussed here. These allogeneic responses might successfully be harnessed to promote the immune eradication of metastatic cancer.

A powerful cooperative interaction between a fusogenic peptide and lipofectamine for the enhancement of receptor-targeted, non-viral gene delivery via integrin receptors.

BACKGROUND: Following receptor-mediated endocytosis, vector/DNA complexes require assistance to exit endocytic vesicles in order to avoid degradation in the lysosomes. Overcoming this barrier is a major challenge for the development of receptor-targeted, non-viral gene delivery. METHODS: The fusogenic peptide of influenza virus haemagglutinin, lipofectamine and chloroquine were tested singly and in combination in various doses for promoting in vitro gene transfer by an integrin-targeted, non-viral DNA vector (polylysine-molossin). RESULTS: The fusogenic peptide and lipofectamine both individually promoted integrin-targeted gene delivery. However, the combined use of these agents was particularly effective, even at concentrations where neither agent singly had any effect on promoting gene delivery by polylysine-molossin. This optimal combination was effective on several cell lines and primary cell cultures. On the HuH7 cell line, it was approximately five-fold more effective than optimal chloroquine concentrations for integrin-targeted gene delivery and four to five times more effective than commercially available polyethylenimine. With the beta-galactosidase reporter gene, 60-65% of HepG2 cells and 75-80% of HuH7 cells were positive. The surface charge of polylysine-molossin/DNA/lipofectamine/fusogenic peptide complexes was approximately the same as that of polylysine-molossin/DNA complexes. The size distribution of the complexes suggested that competitive binding of polylysine-molossin and lipofectamine to DNA influenced the overall efficacy of this approach. CONCLUSIONS: Although the mechanisms are not clear, the combined use of very low doses of two membrane-destabilizing agents results in high levels of receptor-targeted gene delivery.

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.

Specific suppression of interleukin 2 biosynthesis by synthetic antisense oligodeoxynucleotides does not influence allograft rejection.

BACKGROUND: Interleukin (IL)-2 supplementation can reverse both blood transfusion-induced tolerance to kidney allografts and spontaneous tolerance to liver allografts in rats. Moreover, IL-2 expression is frequently suppressed in models of allograft tolerance. The failure of IL-2 biosynthesis might therefore play a critical role in tolerance induction. METHODS: Three antisense oligodeoxynucleotides (AS-1, AS-2, AS-3) to rat IL-2, and a control oligo (C-1) consisting of a scrambled version of AS-1, were evaluated for gene-specific suppression of IL-2 biosynthesis in vitro and in vivo, and for their effects on kidney allograft survival. Reverse transcriptase-polymerase chain reaction and IL-2 protein assays were used to assay concanavalin A-driven IL-2 biosynthesis by lymph node lymphocytes in vitro. PVG recipients of Dark Agouti kidney allografts were treated with the oligos. Graft survival and IL-2 biosynthesis by reverse transcriptase-polymerase chain reaction in spleen and graft biopsy specimens were assessed. RESULTS: The AS-1 oligo, but not the AS-2, AS-3 or C-1 oligos, suppressed concanavalin A-driven IL-2 biosynthesis for the 4 days of culture. This effect was dependent on delivery of the AS-1 oligo with lipofectamine. Supplementation with exogenous IL-2 reversed the suppression of lymphocyte proliferation in AS-1-treated cultures. Administration of AS-1 intravenously at 10 mg/kg/day to PVG recipients of Dark Agouti kidney allografts suppressed IL-2 (but not IL-6, interferon-gamma, or tumor necrosis factor-alpha) synthesis in the grafts of seven of nine rats, as measured in biopsy specimens taken at days 2-7. By contrast, all nine control grafts strongly expressed IL-2. However, neither graft histopathology nor graft survival was affected. CONCLUSIONS: Antisense oligonucleotides can powerfully suppress IL-2 biosynthesis in vitro and in allograft recipients in vivo, but this does not affect kidney allograft rejection.

An epithelial cell line that can stimulate alloproliferation of resting CD4+ T cells, but not after IFN-gamma stimulation.

It has previously been shown that IFN-gamma-induced up-regulation of HLA class II on the surface of epithelial cells is not sufficient to induce proliferation of allospecific CD4+ T cells in vitro. To further investigate this phenomenon, a human epithelial bladder carcinoma, T24, was induced to constitutively express HLA class II without IFN-gamma stimulation, by permanent transfection with the full-length class II transactivator (CIITA) gene. Proliferation of allospecific T cells to transfected and wild-type cells with and without prior activation with saturating levels of IFN-gamma for 4 days was examined. IFN-gamma-activated T24 did not induce any response from CD4+ T cells. However, T24.CIITA induced significant levels of alloproliferation, which could be abrogated by pretreatment of T24.CIITA with a mAb to LFA-3. Prestimulation of T24. CIITA with saturating levels of IFN-gamma for 4 days also prevented allospecific CD4+ T cell proliferation. These findings suggest that epithelial cells may be intrinsically able to process and present alloantigen and provide adequate costimulation. We propose that IFN-gamma has a secondary, as yet unidentified, effect that acts to negatively regulate this response, at least in some epithelial cells.

Tissue-binding properties of a synthetic peptide DNA vector targeted to cell membrane integrins: a possible universal nonviral vector for organ and tissue transplantation.

BACKGROUND: Gene delivery through a nonviral, receptor-mediated system widely expressed in transplanted tissue would have important advantages in transplantation, where gene delivery is performed ex vivo. Integrins are widely expressed cell surface receptors and can be targeted for gene delivery. METHODS: A synthetic 31 amino acid DNA vector (polylysine-molossin) comprising a 15-amino acid moiety for targeting cellular integrins (derived from the snake venom, molossin) and a 16-amino acid polylysine moiety for DNA-binding, has been evaluated. The 31-amino acid vector, as well as its separate 15-amino acid integrin-binding and (lys)16 components, were individually synthesized, and a monoclonal antibody was raised to the molossin peptide for these studies. Binding to cell lines and tissue sections and capacity for gene delivery were examined. RESULTS: Flow cytometric studies with the ECV304 cell line demonstrated that the binding of polylysine-molossin and polylysine-molossin/DNA complexes involved both electrostatic and integrin-mediated interactions with the cells, with the electrostatic binding being sufficient for maximal binding. However, binding to cellular integrins was essential for successful gene transfer. Binding studies on frozen tissue sections of the rat and pig demonstrated that the molossin peptide bound to many cell types of interest in transplantation, but not to all. Among the negative tissues were vascular endothelium and pancreatic islets. Small species differences in tissue binding were noted between the rat and pig. CONCLUSIONS: This study defines the cooperative nature of the binding of this vector system to target cells and establishes the cell types most likely to be effectively targeted for DNA transfer.

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.

Efficient gene delivery to vascular smooth muscle cells using a nontoxic, synthetic peptide vector system targeted to membrane integrins: a first step toward the gene therapy of chronic rejection.

BACKGROUND: Chronic rejection is now the major cause of allograft failure. A prominent characteristic of the histopathology is extensive intimal proliferation of vascular smooth muscle cells. Targeting vascular smooth muscle cells by gene therapy techniques offers a possible avenue for arresting or reversing chronic rejection. Defining suitable non-viral DNA vectors for this application is the objective of this study. METHODS: A 31 amino acid synthetic peptide has been evaluated as a DNA vector for primary cultures of vascular smooth muscle cells of man, rabbit, and rat. The vector comprises a 15 amino acid integrin-binding domain and a chain of 16 lysines for electrostatic binding of DNA. Three agents known to promote exit of vector/DNA complexes from endocytic vesicles were studied systematically to define optimal, non-toxic conditions for gene delivery. RESULTS: Initial binding studies on frozen sections showed that the integrin-binding domain binds strongly to vascular smooth muscle cells in all three species, thereby establishing vascular smooth muscle cells as a potential target for this receptor-targeted DNA vector system. Primary cultures of vascular smooth muscle were therefore studied. The use of chloroquine to assist endocytic exit, which works well on immortalized cell lines, was of little value because of toxicity to the primary vascular smooth muscle cells. The addition of cationic lipids to polylysine-molossin/DNA conjugates gave excellent reporter gene expression, but required mildly toxic doses of cationic lipid, and resulted in some loss of integrin specificity of the vector system. The optimal system involved the use of the amino terminal 20 amino acids of the hemagglutinin of the influenza virus. This peptide, when added to polylysine-molossin/DNA complexes at an optimal w/w ratio of 5:1:2 (polylysine-molossin/DNA/fusogenic peptide) resulted in 25-30% transfection of vascular smooth muscle cells with good levels of gene expression and no toxicity. CONCLUSION: This represents an effective and safe DNA vector, comprised entirely of small synthetic peptides, and therefore readily standardized for clinical and experimental application.

The induction of major histocompatibility complex class II expression is sufficient for the direct activation of human CD4+ T cells by porcine vascular endothelial cells.

BACKGROUND: The role played by major histocompatibility complex (MHC) class II-positive vascular endothelial cells in organ graft rejection is unknown but potentially very important. Methods. The MHC class II-negative porcine vascular endothelial cell line PIEC was stably transfected with the human class II transactivator CIITA, in order to induce MHC class II expression without the coinduction of T-cell costimulatory ligands. These PIEC cells were compared with interferon gamma-treated PIEC cells for their capacity to stimulate the proliferation of pure human CD4+ T cells. Results. The CIITA-transfected PIECs were as effective as interferon y-treated PIECs for stimulating unprimed human CD4+ T cells, the peak response with the CIITA-transfected cells in fact occurring earlier (day 3 instead of day 5). Monoclonal antibodies to SLA-DR substantially inhibited the CD4+ T-cell responses in both cases. However, whereas the response to interferon gamma-treated PIEC was partially inhibited by CTLA4-Ig, that to CIITA-transfected PIEC was not. Conclusions. The strong stimulation of CD4+ T cells by the specific induction of MHC class II antigens demonstrates that PIEC cells constitutively express functionally effective levels of costimulatory ligands. This finding strengthens the case that vascular endothelial cells are professional antigen-presenting cells and that MHC class II-positive vascular endothelial cells might play a role in the rejection of organ allografts.

Suppression of human anti-porcine T-cell immune responses by major histocompatibility complex class II transactivator constructs lacking the amino terminal domain.

BACKGROUND: The class II transactivator (CIITA) is a bi- or multifunctional domain protein that acts as a transcriptional activator and plays a critical role in the expression of MHC class II genes. We have previously demonstrated that a mutated form of the human CIITA gene, coding for a protein lacking the amino terminal 151 amino acids, acts as a potent dominant-negative suppressor of HLA class II expression. Porcine MHC class II antigens are potent stimulators of direct T-cell recognition by human CD4+ T cells and are, therefore, likely to play an important role in the rejection responses to transgenic pig donors in clinical xenotransplantation. We were, therefore, interested in examining mutated CIITA constructs for their effect on porcine MHC class II expression. METHODS: Stable transfectants of the porcine vascular endothelial cell line PIEC with mutated CIITA constructs were tested for SLA-DR and SLA-DQ induction by recombinant porcine interferon-gamma. Transient transfectants of the porcine B-cell line L23 with the mutated CIITA constructs were tested for the suppression of constitutive SLA-DR and SLA-DQ expression. T-cell proliferation studies were performed using highly purified human CD4+ T cells. RESULTS: In preliminary studies, we demonstrated that transfection of the PIEC line with full-length human CIITA constructs resulted in strong expression of SLA-DR and SLA-DQ antigens, thus establishing the cross-species effectiveness of human CIITA in the pig. The mutated human CIITA constructs were, therefore, tested in the pig. PIEC clones stably transfected with one of these constructs showed up to 99% suppression of SLA-DR and SLA-DQ antigen induction and marked suppression of SLA-DRA mRNA induction. Moreover, transient transfection of the porcine B-cell line L23 showed up to 90% suppression of constitutive SLA-DR and SLA-DQ antigen expression in 5-8 days. In functional studies, interferon-gamma-stimulated PIEC clones transfected with this mutated CIITA construct failed to stimulate purified human CD4+ T lymphocytes. CONCLUSION: Mutated human CIITA constructs are potent suppressors of porcine MHC class II expression.

Suppression of MHC class II expression by human class II trans-activator constructs lacking the N-terminal domain.

The class II trans-activator (CIITA) is a bi- or multi-functional domain protein which plays a critical role in the expression of MHC class II genes. We report that removal of the N-terminal 151 amino acids, encompassing all of the acidic domain but leaving intact the proline/serine/threonine-rich domain, results in a mutant protein with potent suppressive properties for MHC class II expression. HeLa cells stably or transiently transfected with mutant CIITA constructs showed up to 99% suppression of MHC class II antigen induction by IFN-gamma and marked suppression of HLA-DRA mRNA expression. Transient transfection of a B lymphoma line resulted in up to 89% reduction of constitutive MHC class II expression within 5 days and suppression of HLA-DRA mRNA synthesis.