Hepatic Transduction Research Articles

Pseudo-hydrodynamic Delivery of Helper-dependent Adenoviral Vectors into Non-human Primates for Liver-directed Gene Therapy

Helper-dependent adenoviral vectors (HDAds) are attractive for liver-directed gene therapy because they can mediate long-term, high-level transgene expression without chronic toxicity. However, systemic delivery requires high vector doses for efficient hepatic transduction, resulting in dose-dependent acute toxicity. Clearly, strategies to improve hepatic transduction with low vector doses are needed. In this regard, we have previously shown that hydrodynamic injection of helper-dependent adenoviral vectors into mice results in increased hepatic transduction, reduced systemic vector dissemination, and reduced pro-inflammatory cytokines compared with conventional injection and thus has the potential to improve dramatically the therapeutic index of helper-dependent adenoviral vectors. Unfortunately, the rapid, large-volume injection used in this method cannot be applied to larger animals. Therefore, we have developed a novel balloon occlusion catheter-based method to mimic hydrodynamic injection of helper-dependent adenoviral vectors into non-human primates that does not require rapid, large-volume injection. Using a low, clinically relevant vector dose, this minimally invasive method results in high-efficiency hepatic transduction with minimal toxicity and stable long-term transgene expression for at least 413 days.

Impairment of hepatic Stat-3 activation and reduction of PPARα activity in fructose-fed rats

Fructose makes up a significant proportion of energy intake in westernized diets; its increased consumption has paralleled the growing prevalence of obesity and metabolic syndrome over the past two decades. In the current study, we demonstrate that fructose administration (10% wt/vol) in the drinking water of rats reduces the trans-activating and trans-repressing activity of the hepatic peroxisome proliferator-activated receptor alpha (PPARalpha). As a consequence, fructose decreases hepatic fatty oxidation and increases pro-inflammatory transcription factor nuclear factor kappaB (NF-kappaB) activity. These changes were not observed in glucose-administered rats (10% wt/vol), although both carbohydrates produced similar changes in plasma adiponectin and in the hepatic expression of transcription factors and enzymes involved in fatty acid synthesis. Fructose-fed, but not glucose-fed, rats were hyperleptinemic and exhibited increased tyrosine phosphorylation of the signal transducer and activator of transcription-3 (STAT-3) transcription factor, although they did not present a similar increase in the serine phosphorylation of nuclear STAT3. Thus, an impairment in the hepatic transduction of the leptin signal could be responsible for the observed alterations in PPARalpha activity in fructose-fed rats. Because PPARalpha activity is lower in human than in rodent liver, fructose ingestion in humans should cause even worse effects, which would partly explain the link between increased consumption of fructose and widening epidemics of obesity and metabolic syndrome. Hypertriglyceridemia and hepatic steatosis induced by fructose ingestion result from a reduction in the hepatic catabolism of fatty acids driven by a state of leptin resistance.

50 Role of the GH/IGF-I Axis in the Growth Retardation of Weaver Mice

IGF-I is an anabolic growth factor essential for growth and development, both as a mediator of growth hormone (GH) action and as a growth factor for cell proliferation and differentiation. Although the role of the GH/IGF-I axis is established for normal postnatal growth, its functional state in neurodegenerative diseases is not fully characterized. The weaver (wv) mutant mouse is a commonly used model for studying hereditary cerebellar ataxia and provides an opportunity to study the function of IGF-I in postnatal growth during neurodegeneration. Previously, we reported that weaver mice are growth retarded and their body weights correlate with a decrease in circulating IGF-I levels, suggesting that IGF-I's endocrine function is impaired in weaver mice. This study further investigated the cause of lower circulating IGF-I levels. Most circulating IGF-I is synthesized in the liver under the control of GH. We found that GH levels in wv mice are reduced, but the hepatic GH receptor signal transduction pathway functions normally, because acute GH treatment increased STAT5b phosphorylation and IGF-I mRNA levels in weaver mice. To investigate whether decreased circulating GH contributes to wv mouse growth retardation, we treated weaver mice with GH for 2 weeks and found a signify cant increase in body weights and circulating IGF-I levels in weaver mice, but not in wild type mice. In summary, our results suggest that postnatal growth retardation in weaver mutant mice likely results from a dysfunction in the GH/IGF-I axis.

Improved Hepatic Transduction, Reduced Systemic Vector Dissemination, and Long-Term Transgene Expression by Delivering Helper-Dependent Adenoviral Vectors into the Surgically Isolated Liver of Nonhuman Primates

Helper-dependent adenoviral vectors (HDAds) are attractive vectors for liver-directed gene therapy because they can mediate sustained, high-level transgene expression without chronic toxicity. However, high vector doses are required to achieve efficient hepatic transduction by systemic delivery because of a nonlinear dose response. Unfortunately, such high doses result in systemic vector dissemination and dose-dependent acute toxicity with potentially severe and lethal consequences. We hypothesize that the threshold to efficient hepatic transduction may be circumvented by delivering the vector into the surgically isolated liver via the portal vein. Total hepatic isolation was achieved by occluding hepatic inflow from the portal vein and hepatic artery and by occluding hepatic venous outflow at the inferior vena cava. We demonstrate in nonhuman primates that this approach resulted in significantly higher efficiency hepatic transduction with reduced systemic vector dissemination compared with systemic intravascular delivery. This method of delivery was associated with transient acute toxicity, the severity of which was variable. Importantly, stable, high levels of transgene expression were obtained for at least 665 days for one baboon and for at least 560 days for two baboons with no evidence of long-term toxicity.

Improved cardiac gene transfer by transcriptional and transductional targeting of adeno-associated viral vectors

Vectors based on recombinant adeno-associated virus 2 (AAV-2) are a promising tool for cardiac gene transfer. However, potential therapeutic applications need to consider the predominant transduction of the liver once AAV-2 vectors enter the systemic circulation. We therefore aimed to increase efficiency and specificity of cardiac vector delivery by combining transcriptional and cell surface targeting. For analysis of transcriptional targeting, recombinant AAV vectors were generated harboring a luciferase reporter gene under control of the cytomegalovirus (CMV) promoter or the 1.5-kb cardiac myosin light chain promoter fused to the CMV immediate-early enhancer (CMV(enh)/MLC1.5). Luciferase activities were determined in representative organs three weeks after intravenous injection of the vector into adult mice. Transductional targeting was studied using luciferase-reporter constructs crosspackaged into capsids of AAV serotypes 1 to 6 and modified AAV-2 capsids devoid of binding their primary receptor heparan sulfate proteoglycan. Intravenous injections of AAV-2 vectors harboring the CMV(enh)/MLC1.5 promoter enabled a specific and 50-fold higher reporter gene expression in left ventricular myocardium of adult mice compared to vectors containing the CMV promoter. Comparison of AAV-2 vector genomes crosspackaged into capsids of AAV-1 to -6 showed that AAV-1, -4, -5, and -6 capsids increased cardiac transduction efficiency by about 10-fold. However, transduction of other organs such as the liver was also increased after systemic administration. In contrast, AAV-2-based vectors with ablated binding to their primary receptor heparan sulfate proteoglycan enabled a significantly increased efficiency of cardiac gene transfer and reduced transduction of the liver. Combining transcriptional targeting by the CMV(enh)/MLC1.5 promoter and AAV vectors devoid of binding the AAV-2 primary receptor results in an efficient cardiac gene transfer with a significantly reduced hepatic transduction.

Ciliary Neurotrophic Factor Prevents Acute Lipid-Induced Insulin Resistance by Attenuating Ceramide Accumulation and Phosphorylation of c-Jun N-Terminal Kinase in Peripheral Tissues

Ciliary neurotrophic factor (CNTF) is a member of the gp130 receptor cytokine family recently identified as an antiobesity agent in rodents and humans by mechanisms that remain unclear. We investigated the impact of acute CNTF treatment on insulin action in the presence of lipid oversupply. To avoid confounding effects of long-term high-fat feeding or genetic manipulation on whole-body insulin sensitivity, we performed a 2-h Intralipid infusion (20% heparinized Intralipid) with or without recombinant CNTF pretreatment (Axokine 0.3 mg/kg), followed by a 2-h hyperinsulinemic-euglycemic clamp (12 mU/kg.min) in fasted, male Wistar rats. Acute Intralipid infusion increased plasma free fatty acid levels from 1.0 +/- 0.1 to 2.5 +/- 0.3 mM, which subsequently caused reductions in skeletal muscle (insulin-stimulated glucose disposal rate) and liver (hepatic glucose production) insulin sensitivity by 30 and 45%, respectively. CNTF pretreatment completely prevented the lipid-mediated reduction in insulin-stimulated glucose disposal rate and the blunted suppression of hepatic glucose production by insulin. Although lipid infusion increased triacylglycerol and ceramide accumulation and phosphorylation of mixed linage kinase 3 and c-Jun N-terminal kinase 1 in skeletal muscle, CNTF pretreatment prevented these lipid-induced effects. Alterations in hepatic and muscle insulin signal transduction as well as phosphorylation of c-Jun N-terminal kinase 1/2 paralleled alterations in insulin sensitivity. These data support the use of CNTF as a potential therapeutic means to combat lipid-induced insulin resistance.

108. Study of an AAV-8 Capsid Mutant with Direct Heparin Binding Capability but Reduced Efficiency in Liver-Targeted Transduction

Top of pageAbstract AAV vectors pseudotyped with the AAV-8 capsids have become a popular alternative to the more extensively studied AAV-2 vectors due to their more robust transduction of liver and other targets in vivo. Importantly, liver transduction with AAV-2 is significantly higher when the vector is administered into the liver vasculature compared to a peripheral venous infusion (e.g. tail vein). In contrast, there are no differences in liver transduction efficiencies when AAV- 8 vectors were administered via these two different routes. Despite the high degree of sequence homologies (|[sim]|83%), the two capsids differ in their cell surface binding properties. Because wild type AAV-8 cannot directly bind to heparin, whose analog, heparan sulfate proteoglycan (HSPG), is a primary cell surface receptor for AAV-2, we hypothesized that the heparin binding motif may play a role in the observed differences in the hepatic transduction parameters. Sequence alignment revealed that the AAV-8 cap gene lacks two basic amino acid residues found in AAV-2 (R585, R588) that are essential for heparin binding. By substituting the corresponding AAV-8 residues at these and neighboring positions with AAV-2 sequence, we generated a mutant AAV-8 (mAAV-8) capsid with direct heparin-binding affinity similar to that of AAV-2. We made liver-specific human factor IX (hFIX)-expressing vectors in AAV-2, AAV-8 or the mAAV-8 capsids respectively, and compared their liver transduction efficiencies following intravenous administration into C57BL/6 mice. Depending on the route of injection, hFIX expression levels from the mAAV-8 vector were three and ten times lower than that of AAV-8 when administered via portal vein and tail vein, respectively. These differences in transduction with peripheral and liver vascular infusions were similar to what we observed with AAV-2, suggesting that the heparin binding motif may influence differing transduction efficiencies when administered by different vascular routes. To further establish this possibility, vector bio-distribution in various tissues was also checked by real-time PCR quantitation of viral genomes at different time points following vector administration. We found that the mAAV-8 capsid showed broader tropism and less targeting in the liver when administered via tail vein. Further experiments are being carried out to examine the intracellular trafficking pathways of vectors made with each of these capsids but our results suggest that vector biodistribution and perhaps intracellular transduction pathways are influenced by the presence of the heparin binding motif.

213. Improving the Therapeutic Index of Helper-Dependent Adenoviral Vector for Crigler-Najjar Gene Therapy

Crigler|[ndash]|Najjar syndrome is an inborn error of metabolism characterized by severe unconjugated hyperbilirubinemia caused by a deficiency of uridine diphospho-glucuronosyl transferase 1A1 (UGT1A1). Current treatment relies on phototherapy to prevent kernicterus, and liver transplantation to prevent long term complications. Life-long correction of hyperbilirubinemia by liver directed gene therapy using a helper-dependent adenoviral vector (HDAd) has been recently reported in the Gunn rat model. However, in that study complete phenotypic correction was only achieved using a dose of |[ge]|3x1012 vp/kg and only partial correction was observed at 6x1011 vp/kg. These doses are very high and may be lethal in humans. To increase the therapeutic index, we injected Gunn rats with a HDAd bearing an improved expression cassette containing the PEPCK promoter, the hUGT1A1 cDNA, the WPRE and a liver specific enhancer (LCR). We observed complete phenotypic correction with 5x1011 vp/kg and partial correction with 1x1011 vp/kg. These are the lowest therapeutic doses reported to date. We have previously shown that hydrodynamic injection of HDAd results in increased hepatic transduction compared to conventional injection. Combining this method of delivery with our improved construct, complete correction was achieved with 5x1010 vp/kg for the duration of the observation period (at least 6 months). Although hydrodynamic injection is not feasible in large animals, we have developed methods to mimic hydrodynamic injection of HDAd into larger animals which have yielded encouraging results (see accompanying abstract). Taken together these strategies may offer clinical gene therapy for Crigler|[ndash]|Najjar syndrome.

1087. Balloon Occlusion Catheter-Based Delivery of HDAd into the Nonhuman Primate Liver Results in Stable, High Level Transgene Expression with Minimal Toxicity

Helper-dependent adenoviral vectors (HDAds) hold tremendous potential for liver-directed gene therapy because they can mediate long-term transgene expression without chronic toxicity. However, due to a nonlinear dose-response, high doses are required to achieve efficient hepatic transduction resulting in dose-dependent acute toxicity. To overcome this important obstacle, we have developed a minimally invasive method to preferentially deliver low dose HDAd into the liver of 30 kg baboons to achieve efficient hepatic transduction. Briefly, a single sausage-shaped balloon occlusion catheter was percutaneously positioned in the inferior vena cava (IVC) of baboon 1 to occlude hepatic venous outflow (Fig. 1A). 1x1011 vp/kg of a HDAd expressing the baboon alpha-fetoprotein (bAFP) marker was injected directly into the occluded liver via a percutaneously placed hepatic artery (HA) catheter and left to dwell within the liver for 15 min before balloon deflation. As controls, 1x1011 vp/kg was administered to baboon 2 by peripheral intravenous injection and baboon 3 by HA injection without balloon occlusion. All procedures were uneventful, well tolerated, and following recovery from anesthesia, all three animals returned to their normal, active pre-injection states with no clinical manifestations of toxicity. Mild transaminitis was observed for all animals, peaking at 24 to 48 h post-vector but returning towards baseline the next day: For ALT, a 2.3, 1.1, and 1.4-fold increase over baseline were observed for baboons 1, 2 and 3, respectively. For AST, a 4.8, 1.6 and 2.8-fold increase were observed for baboons 1, 2 and 3, respectively. Importantly, serum bAFP levels increased 425-fold over baseline for baboon 1 (from 4.5 ng/ml at a baseline to 1914 ng/ml), while only a modest increase of 12-fold and 5.6-fold were observed for baboon 2 (from 9 ng/ml to 109 ng/ml) and baboon 3 (from 18 ng/ml to 103 ng/ml), respectively (Fig. 1B). To date, these bAFP levels have been sustained (at least 56 days). These results suggest that the therapeutic index of HDAd can be significantly improved by delivering the vector preferentially into the liver using a minimally invasive balloon occlusion catheter technique and may be a first step towards clinical application of HDAd for liver-directed gene therapy.

In vivo hepatic HB-EGF gene transduction inhibits Fas-induced liver injury and induces liver regeneration in mice: A comparative study to HGF

It is unknown whether heparin-binding EGF-like growth factor (HB-EGF) can be a therapeutic agent, although previous studies suggested that HB-EGF might be a hepatotrophic factor. This study explores the potential of hepatic HB-EGF gene therapy in comparison with HGF. Mice received an intraperitoneal injection of the agonistic anti-Fas antibody 72 h after an intravenous injection of either adenoviral vector (1x10(11) particles) expressing human HB-EGF (Ad.HB-EGF), human HGF (Ad.HGF) or no gene (Ad.dE1.3), and were sacrificed 24 or 36 h later to assess liver injury and regeneration. Exogenous HB-EGF was predominantly localized on the membrane, suggesting the initial synthesis of proHB-EGF in hepatocytes. The control Ad.dE1.3-treated mice represented remarkable increases in serum ALT and AST levels and histopathologically severe liver injuries with numerous apoptosis, but a limited number of mitogenic hepatocytes. In contrast, the liver injuries and apoptotic changes were significantly inhibited, but the mitogenic hepatocytes remarkably increased, in both the Ad.HB-EGF- and Ad.HGF-treated mice. More mitogenic hepatocytes and milder injuries were observed in the Ad.HB-EGF-treated mice. HB-EGF has more potent protective and mitogenic effects for hepatocytes than HGF, at least for the present conditions. In vivo hepatic HB-EGF gene transduction is therapeutic for Fas-induced liver injury.

Efficient Hepatic Delivery and Expression from a Recombinant Adeno-associated Virus 8 Pseudotyped α1-Antitrypsin Vector

α1-Antitrypsin (AAT) deficiency is a single-gene disorder in which a mutation in the AAT (approved symbol SERPINA1) gene (PI*Z) leads to misfolding of the protein, loss of the protective antiprotease effect of AAT for the lungs, and a toxic effect on hepatocytes. Optimal therapy for AAT deficiency will require a high percentage of hepatocyte transduction to be effective for liver and lung disease. Recently, rAAV genomes pseudotyped with capsids from serotypes 7 and 8 showed efficient hepatic transduction. We hypothesized that upon portal vein injection to target hepatocytes, serotype 8 would better transduce target cells and therefore express hAAT in both a greater percentage of cells and greater amounts. AAV2 and pseudotyped vectors for serotypes 1, 5, and 8 carrying the human AAT transgene were injected at 1 × 1010 particle doses into C57Bl/6 mice. Circulating hAAT from AAV2/8-injected animals showed a 2-log advantage over AAV2 and 3-log increase over AAV2/1 and 5 for the 24-week study. Most significantly, up to 40% of total liver cells stained positive for the transgene in AAV2/8 subjects while remaining primarily episomal. Therefore, pseudotyped AAV8 provides a vehicle to infect a high percentage of hepatocytes stably and thereby express therapeutic molecules to modify AAT PiZ transcripts.

Influence of Fiber Detargeting on Adenovirus-Mediated Innate and Adaptive Immune Activation

The major adenovirus (Ad) capsid proteins hexon, penton, and fiber influence the efficiency and tropism of gene transduction by Ad vectors. Fiber is the high-affinity receptor binding protein that serves to mediate cell attachment in vitro when using coxsackie-adenovirus receptor (CAR)-containing cell lines. This contrasts with transduction efficiency in macrophages or dendritic cells that lack high concentrations of CAR. To determine how fiber influences gene transduction and immune activation in a murine model, we have characterized Ad type 5 (Ad5) vectors with two classes of chimeric fiber, CAR binding and non-CAR binding. In a systemic infection, Ad5 fiber contributes to DNA localization and vector transduction in hepatic tissue. However, the majority of vector localization is due to Ad5 fiber-specific functions distinct from CAR binding. CAR-directed transduction occurs but at a modest level. In contrast to CAR binding vectors, the F7 and F7F41S non-CAR-binding vectors demonstrate a 2-log decrease in hepatic transduction, with a 10-fold decrease in the amount of vector DNA localizing to the hepatic tissue. To characterize the innate response to early infection using fiber chimeric vectors, intrahepatic cytokine and chemokine mRNAs were quantified 5 hours postinfection. Tumor necrosis factor alpha mRNA levels resulting from Ad5 fiber infections were elevated compared to viruses expressing serotype 7 or 41 fiber. Levels of chemokine mRNA (gamma interferon-inducible protein 10, T-cell activation gene 3, and macrophage inflammatory protein 1beta) were 10- to 20-fold higher with CAR binding vectors (Ad5 and F41T) than with non-CAR-binding vectors (F7 and F7F41S). In spite of quantitative differences in vector localization and innate activation, fiber pseudotyping did not significantly change the outcome of anti-Ad adaptive immunity. All vectors were cleared with the same kinetics as wild-type Ad5 vectors, and each induced neutralizing antibody. Although non-CAR-binding vectors were impaired in transduction by nearly 2 orders of magnitude, the level of antitransgene immunity was the same for each of the vectors. Using primary bone marrow-derived macrophages and dendritic cells, we demonstrate that transduction, induction of cytokine/chemokine, and phenotypic maturation of these antigen-presenting cells are independent of fiber content. Our data support a model where fiber-mediated hepatic localization enhances innate responses to virus infection but minimally impacts on adaptive immunity.

Increased Hepatic Transduction with Reduced Systemic Dissemination and Proinflammatory Cytokines Following Hydrodynamic Injection of Helper-Dependent Adenoviral Vectors

Hydrodynamic injection of helper-dependent adenoviral vectors (HDAd) in mice results in increased hepatic transduction, reduced splenic and pulmonary transduction, and reduced levels of the proinflammatory cytokines IL-6 and IL-12 compared to conventional injection. These results indicate that hepatic transduction by HDAd, at least alone, does not necessarily provoke a severe innate inflammatory response. Instead, they suggest that systemic vector dissemination may play a major role in the severity of the innate inflammatory response. These results further suggest that the safety and efficacy of HDAd-mediated, liver-directed gene therapy may be improved if the vector could be preferentially, if not exclusively, targeted to liver.

906. High Efficiency Hepatic Transduction with Minimal Toxicity Following “Hydrodynamic” Injection of HDAd into Nonhuman Primates

Helper-dependent adenoviral vectors (HDAd) are attractive vectors for liver-directed gene therapy because they can mediate sustained, high level transgene expression with no long-term toxicity. However, as a consequence of a threshold effect, high vector doses are required to achieve efficient hepatic transduction by peripheral intravenous injection which unfortunately results in a dose-dependent activation of the innate inflammatory response. Clearly strategies to overcome the threshold to efficient hepatic transduction are needed to improve the therapeutic index of HDAd. We have recently shown that systemic hydrodynamic injection of HDAd into mice resulted in exceedingly high levels of hepatic transduction and reduction in both systemic vector dissemination and acute proinflammatory cytokines compared to conventional injection. Hydrodynamic injection offers a method of dramatically increasing the therapeutic index of HDAd but, unfortunately, cannot be applied to larger animals due to the large injection volume. Therefore, we have developed a minimally invasive method to mimic hydrodynamic injection in nonhuman primates which does not require injection of large volumes. This method involves the use of balloon occlusion catheters percutaneously positioned in the vena cava to transiently obstruct hepatic venous outflow to increase the intrahepatic pressure prior to simple peripheral intravenous injection of HDAd in a small volume. We demonstrate in nonhuman primates that this novel method of vector delivery results in an exceedingly high and unprecedented level of hepatic transduction with low vector doses resulting in stable, long-term transgene expression. This was accompanied by reduced systemic vector dissemination and minimal, transient elevations of proinflammatory cytokines. Despite the relatively high intrahepatic pressures achieved, there was minimal evidence of liver injury; no histological abnormalities were observed and only minimal and transient elevation of AST and ALT were noted. The procedure itself was uneventful and very well tolerated with no clinical manifestations of acute or chronic toxicities. This novel method of delivering HDAd is simple, minimally invasive, and clinically relevant since it increases the therapeutic index of HDAd for liver-directed gene therapy by allowing high efficiency hepatic transduction with low vector doses and with negligible acute or chronic toxicities.

904. High Efficiency Hepatic Transduction and Long-Term Transgene Expression by Delivering Helper-Dependent Adenoviral Vectors into the Surgically Isolated Liver of Nonhuman Primates

Helper-dependent adenoviral vectors (HDAd) are attractive vectors for liver-directed gene therapy because they can mediate long-term, high level transgene expression with no long-term toxicity. However, as a consequence of a threshold effect, high vector doses are required to achieve efficient hepatic transduction by peripheral intravenous injection which unfortunately results in a dose-dependent activation of the innate inflammatory response. Clearly strategies to overcome the threshold to efficient hepatic transduction are needed to improve the therapeutic index of HDAd. We hypothesized that this obstacle could be surmounted by delivering the vector exclusively to the liver. To test this hypothesis, we have injected HDAd directly into the surgically isolated liver via the portal vein in nonhuman primates. Total hepatic isolation was achieved by occluding hepatic inflow from the portal vein and hepatic artery and by occluding hepatic venous outflow at the inferior vena cava. Prior to total hepatic isolation, saline was infused into the portal vein to flush blood out of the liver. The vector was then injected directly into the liver via the portal vein and allowed to dwell for 30 minutes, following which unabsorbed vector was flushed out via a catheter placed in the vena cava to minimize systemic vector dissemination. Our results revealed that significantly higher hepatic transduction efficiencies can be achieved with relatively low vector doses compared to peripheral intravenous injection. Importantly, stable, high levels of transgene expression were observed for up to one year with no long-term toxicity. This approach may increase the safety and efficacy of HDAd-mediated, liver-directed gene therapy by minimizing the dose required to achieve efficient hepatic transduction and by minimizing systemic vector dissemination.

905. Hydrodynamic Injection of Helper-Dependent Adenoviral Vectors Increases Liver Transduction Efficiency and Decreases Acute Inflammatory Response

Hydrodynamic injection of helper-dependent adenoviral vectors (HDAd) in mice results in increased hepatic transduction resulting in higher levels of long-term transgene expression compared to conventional injection. Furthermore, hydrodynamic injection resulted in reduced splenic and pulmonary transduction and reduced serum levels of the proinflammatory cytokines IL-6 and IL-12. Interestingly, hydrodynamic injection does not appear to alter Kupffer cell uptake of the vector compared to conventional injection. Taken together, these results strongly suggest that hepatic transduction by HDAd, at least alone, does not necessarily provoke a severe innate inflammatory response, but that systemic vector dissemination may play a major role in the severity of the innate inflammatory response. These results further suggest that the safety and efficacy of HDAd-mediated, liver-directed gene therapy may be improved if the vector could be preferentially, if not exclusively, targeted to hepatocytes.

908. Evaluation of the Sleeping Beauty Transposon System in OTC Deficient Spf-Ash Mice: A Non-Viral Approach to a Cell Autonomous Metabolic Disease

relatively high intrahepatic pressures achieved, there was minimal evidence of liver injury; no histological abnormalities were observed and only minimal and transient elevation of AST and ALT were noted. The procedure itself was uneventful and very well tolerated with no clinical manifestations of acute or chronic toxicities. This novel method of delivering HDAd is simple, minimally invasive, and clinically relevant since it increases the therapeutic index of HDAd for liver-directed gene therapy by allowing high efficiency hepatic transduction with low vector doses and with negligible acute or chronic toxicities.

Human Factor IX Expression and Hemophilia Phenotypic Correction in Mice: A Comparison of AAV Serotypes.

Recombinant adeno-associated virus (rAAV) has been extensively studied as a vector for hemophilia gene transfer. AAV serotype 2 targeting the liver and skeletal muscle has been used in clinical trials for hemophilia B patients. Previously we reported that the use of recombinant non-AAV2 serotype vectors generated persistent expression of supra-normal levels of canine factor IX (cFIX) in immunodeficient mice (Mol Ther ‘2000), and resulted in sustained and complete hemophilia B phenotype correction in immune competent hemophilia B mice (Mol Ther ‘2001). In those studies AAV serotypes 1–5 were tested by intramuscular injection. In this study we tested rAAV serotypes 1–8 for hepatic transduction and FIX production in both C57BL/6 and FIX knockout mice. Animals received intrapotal vein injection of 1x1011 virion particles of rAAV 1 thru 8 carrying a human FIX cDNA linked to a chicken beta actin enhancer-CMV promoter and bovine growth hormone polyA. Animals were followed for 8 months and assessed for plasma levels of human FIX by ELISA. A rapid, sustained and maximal rate of hFIX expression was observed within 1–2 weeks with all vectors except AAV2 where the peak expression occurred at 8–10 weeks. hFIX expression was sustained for all serotyped vectors (n=5 animals tested at each AAV serotype). Animals that received serotypes 7 and 8 maintained hFIX at physiological levels (100% hFIX ~ 5000 ng/ml). A differential hFIX expression pattern emerged with rAAV7 (6213 ng/ml) > 8 (5111 ng/ml) > 5 (2367 ng/ml) > 1 (1090 ng/ml) > 4 (377 ng/ml) > 2 (314 ng/ml) > 3 (232 ng/ml). rAAV7 and rAAV8 generated 20 times more hFIX per virion particle than the rAAV2 vector. We did not detect anti-human FIX antibody in any of the experimental mice. To assess why hFIX production differed between each serotype we performed immunohistochemical staining of the mouse liver using a fluorescent-tagged anti-human FIX antibody. Based on this assay, rAAV7 and AAV8 transduced 30% of hepatocytes while rAAV2 transduced less than 2% of the hepatocytes. We subsequently tested FIX production and effect on hemophilia phenotype of rAAV 7 and 8 (1x1011 virion particles) in hemophilia B mice; the data was similar to that observed using the C57Bl/6 mice. Human FIX levels (>5000 ng/ml) were again sustained during the 6–8 month observation period. No anti-human FIX antibody was detected in these hemophilia B mice. Results of clotting function by aPTT testing demonstrated a normalization of clotting time in all the rAA8/hFIX treated hemophilia mice. Survival of all treated mice by tail clip (lethal for all non-treated knockout animals) confirmed that phenotypic correction had been achieved. Our results again demonstrate that AAV serotypes have differential transduction rates and that new serotypes with greater mouse hepatocyte transduction rates are more efficient for hemophilia gene transfer.

Relationship between isolated hepatic cholinergic denervation and insulin signal transduction in rat hepatocytes

目的 通过研究去肝脏胆碱能神经(IHCD)大鼠肝细胞胰岛素信号转导过程中信号蛋白表达的改变,探讨IHCD对胰岛素信号转导的影响机制.方法选用健康Wistar大鼠30只, 随机分为手术组、术后给药组和假手术组,每组10只.手术组建立IHCD大鼠模型,术后给药组每日每只动物予罗格列酮0.08 mg灌胃一次,假手术对照.4周后处死实验大鼠,留取肝组织标本,用Western blot及免疫组织化学方法观察手术组大鼠胰岛素受体(IR)、胰岛素受体底物-1(IRS-1)、葡萄糖载体蛋白2(Glut2)等胰岛素信号蛋白及效应蛋白表达的改变,以及药物处理对这些蛋白表达的影响.结果 Western blot显示手术组大鼠肝组织内IR、IRS-1、Glut2蛋白表达较假手术组明显降低(P 0.05),免疫组化的检查结果基本与之一致.结论 IHCD导致胰岛素信号转导通路中IR、IRS-1、Glut2等蛋白的表达降低,从而影响葡萄糖代谢过程,罗格列酮可增加它们的表达,肝脏胆碱能神经的完整性对调节和维持葡萄糖代谢的平衡具有一定的作用。

789. Hydrodynamic Injection of Helper Dependent Adenoviral Vectors Increases Liver Transduction Efficiency and Decreases Acute Inflammatory Response

Top of pageAbstract Hydrodynamic injection of helper–dependent adenoviral vectors (HDAd) in mice results in significantly higher efficiency hepatic transduction and significantly higher levels of transgene expression compared to conventional injection without compromising duration of expression. Furthermore, significantly less splenic transduction was observed following hydrodynamic injection compared to conventional injection. These results suggest that hydrodynamic injection alters vector biodistribution to favor transduction of the liver and consequently reduce transduction of other organs. Moreover, elevations in IL–6 and IL–12 were less following hydrodynamic than conventional injection suggesting that activation of the innate inflammatory response may be reduced by minimizing systemic exposure/transduction through preferential hepatic transduction. Although systemic hydrodynamic injection is not possible in large animals, localized hydrodynamic injection may provide a method of achieving efficient HDAd–mediated hepatic transduction and minimizing the acute inflammatory immune response.