Efficient Hepatic Transduction Research Articles

Correction of Hyperbilirubinemia in Gunn Rats by Surgical Delivery of Low Doses of Helper-Dependent Adenoviral Vectors

Helper-dependent adenoviral (HDAd) vectors are attractive for liver-directed gene therapy because they can drive sustained high levels of 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 potential lethal consequences. We have previously shown in nonhuman primates that delivery of HDAd in surgically isolated livers resulted in a significantly higher hepatic transduction with reduced systemic vector dissemination compared with intravenous delivery and multiyear transgene expression. Encouraged by these data, we have now employed a surgical vector delivery method in the Gunn rat, an animal model for Crigler-Najjar syndrome. After vector delivery into the surgically isolated liver, we show phenotypic correction at the low and clinically relevant vector dose of 1 × 10(11) vp/kg. Correction of hyperbilirubinemia and increased glucuronidation of bilirubin in bile was achieved for up to 1 year after vector administration. Surgical delivery of the vector was well tolerated without signs of acute or chronic toxicity. This method of delivery could thereby be a safer alternative to liver transplantation for long-term treatment of Crigler-Najjar syndrome type I.

Efficient, Long-term Hepatic Gene Transfer Using Clinically Relevant HDAd Doses by Balloon Occlusion Catheter Delivery in Nonhuman Primates

Helper-dependent adenoviral vectors (HDAd) are devoid of all viral coding sequences and are thus an improvement over early generation Ad because they can provide long-term transgene expression in vivo without chronic toxicity. However, high vector doses are required to achieve efficient hepatic transduction by systemic intravenous injection, and this unfortunately results in dose-dependent acute toxicity. To overcome this important obstacle, we have developed a minimally invasive method to preferentially deliver HDAd into the liver of nonhuman primates. Briefly, a balloon occlusion catheter was percutaneously positioned in the inferior vena cava to occlude hepatic venous outflow. HDAd was injected directly into the occluded liver via a percutaneously placed hepatic artery catheter. Compared to systemic vector injection, this approach resulted in substantially higher hepatic transduction efficiency using clinically relevant low vector doses and was accompanied by mild-to-moderate acute but transient toxicities. Transgene expression was sustained for up to 964 days. These results suggest that our minimally invasive method of delivery can significantly improve the vector's therapeutic index and may be a first step toward clinical application of HDAd for liver-directed gene therapy.

986. Balloon Occlusion Catheter-Based Delivery of HDAd Permits Efficient Hepatic Transduction Using Clinically Relevant Low Vector Doses in Nonhuman Primates

986. Balloon Occlusion Catheter-Based Delivery of HDAd Permits Efficient Hepatic Transduction Using Clinically Relevant Low Vector Doses in Nonhuman Primates

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.

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.

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.

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.

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.

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.

862. Improved Efficacy of Adenovirus-Mediated Gene Therapy for GSD-II Disease by Selective Depletion of Kupffer Cells

Glycogen storage disease type II (GSD-II) is a fatal genetic muscle disorder caused by a massive accumulation of glycogen in multiple muscle groups, and is caused by the deficiency of intralysosomal acid alpha glucosidase (GAA). Two approaches to the treatment of GSD-II are currently being pursued in our research group: enzyme replacement therapy and gene therapy. Relative to gene therapy, our previous work demonstrated that a single intravenous administration of advanced generation, [E1-,pol-] adenoviral (Ad) based vectors encoding human GAA (hGAA) resulted in efficient hepatic transduction and secretion of high levels of hGAA, consequently, the correction of all affected muscles in a GAA-KO mouse model as well a quail model of GSD-II occurred. The long-term persistence of the improved vector in liver tissues facilitated significant glycogen reduction in multiple muscles (though, with variable responses in extent and duration among the individual muscle group analyzed) for at least 6 months, despite the fact that hGAA is perceived as a foreign antigen in these animal models. Unfortunately, our most recent work suggests that very high doses of Ad vectors must be intravenously injected to facilitate the large amounts of hepatic hGAA secretion required to correct both cardiac and skeletal muscles in these animal models. To determine if this problem can be overcome, we have begun attempts to pre-emptively suppress the innate immune systems of immunocompetent GAA-KO mice, prior to injection with our viral vectors. Macrophages, in particular hepatic Kupffer cells, play a central role in sequestering Ad vectors, as well in triggering early innate immune responses, and hence have a profound impact on the adaptive immune responses following adenoviral vector administration. In this study we evaluated the effect of selective and transient depletion of Kupffer cells (via pretreatment of mice with clodronate liposomes-dichloromethylene-bisphosphonate) one day prior to vector injection. We intravenously injected into the clodronate liposome pretreated GAA-KO mice different dosages of the vector, to determine how removal of Kupffer cells impacted upon the known efficacy of this approach. We found that clodronate pretreatment allowed lower doses of the vector to show greater efficacy. For example, the GAA activities in the clodronate-treated mice were significantly elevated both in plasma and in tissues such as liver, heart, diaphram and quadriceps, as compared to the non-Kupffer cell depleted mice injected with identical doses of the [E1-,pol-]AdhGAA vector. As a result, the pathogenic glycogen accumulations in clodronate liposome pretreated GAA-KO mice were significantly reduced at lower doses of the Ad vector (10–100 fold less) as compared to Ad treated GAA-KO mice that had not been pretreated with clodronate containing liposomes. Our data also suggests however, that liver toxicities were also now apparent at lower vector dosages, suggesting that Kupffer cells act to prevent liver damage by sequestering Ad particles. Whether or not other “Ad induced” toxicities are prevented by Kupffer cell depletion are studies currently ongoing in our laboratories.

173. Adenovirus-Mediated Gene Transfer to Mouse and Dog Pancreas In Vivo

Helper-dependent adenoviral vectors (HDAd) are attractive vectors for liver-directed gene therapy because they can mediate sustained, high level transgene expression with negligible long-term toxicity. However, high vector doses are required to achieve efficient hepatic transduction by peripheral intravenous injection due to a nonlinear dose response. Unfortunately, such high systemic doses result in wide spread vector dissemination and dose-dependent activation of the acute inflammatory response which can result in severe and lethal acute toxicity. We hypothesize that this problem can be surmounted by delivering the vector exclusively to the liver thereby permitting high efficiency hepatic transduction with low vector doses and with minimal systemic vector dissemination. To test this hypothesis, we injected HDAd directly into the surgically isolated liver via the portal vein in nonhuman primates. Total hepatic isolation was achieved by occluding hepatic blood inflow from the portal vein and hepatic artery and by occluding hepatic blood outflow at the 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 isolated liver via the portal vein and allowed to dwell for 30 min to prolong exposure to hepatocytes. Following the 30 min dwell time, unabsorbed vector was removed from the liver to minimize systemic dissemination. This was accomplished by infusing saline into the portal vein and collecting the unabsorbed vector from a vena cava catheter. We investigated the safety, feasibility and efficacy of this approach and our results revealed that the surgical procedure was well tolerated in nonhuman primates and that significantly higher hepatic transduction efficiencies can be achieved with relatively low vector doses as compared to those obtained by peripheral intravenous injection. 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.

Systemic correction of the muscle disorder glycogen storage disease type II after hepatic targeting of a modified adenovirus vector encoding human acid-alpha-glucosidase.

This report demonstrates that a single intravenous administration of a gene therapy vector can potentially result in the correction of all affected muscles in a mouse model of a human genetic muscle disease. These results were achieved by capitalizing both on the positive attributes of modified adenovirus-based vectoring systems and receptor-mediated lysosomal targeting of enzymes. The muscle disease treated, glycogen storage disease type II, is a lysosomal storage disorder that manifests as a progressive myopathy, secondary to massive glycogen accumulations in the skeletal and/or cardiac muscles of affected individuals. We demonstrated that a single intravenous administration of a modified Ad vector encoding human acid alpha-glucosidase (GAA) resulted in efficient hepatic transduction and secretion of high levels of the precursor GAA proenzyme into the plasma of treated animals. Subsequently, systemic distribution and uptake of the proenzyme into the skeletal and cardiac muscles of the GAA-knockout mouse was confirmed. As a result, systemic decreases (and correction) of the glycogen accumulations in a variety of muscle tissues was demonstrated. This model can potentially be expanded to include the treatment of other lysosomal enzyme disorders. Lessons learned from systemic genetic therapy of muscle disorders also should have implications for other muscle diseases, such as the muscular dystrophies.

Viral IL-10 gene therapy inhibits TNF-α and IL-1β, not IL-6, in the newborn endotoxemic mouse

Introduction: Modulation of the inflammatory cascade within the liver of critically ill infants may improve the chance of survival. Using gene therapy, the authors hypothesized that augmented local production of the counter-regulatory cytokine viral interleukin-10 (IL-10) in vivo will modulate the critical cytokines in the inflammatory response. The purpose of the present study was to determine whether replication-defective adenovirus-mediated viral IL-10 (vIL-10) gene transfer and expression within the liver can achieve this goal in newborn mice. Materials and Methods: Four-week-old Balb c mice were administered (intraperitoneally) 1 × 10 9 plaque-forming units (pfu) per milliliter of an adenovirus vector ( E1a b -deleted ) that encodes the sv40 promoter and the BCRF1 cDNA, or of control vector dl434 that expresses no foreign gene. Forty-eight hours later the mice were challenged with 50 μg/kg of lipopolysaccharide; (LPS) they were killed 1, 2, 6, or 24 hours later (six at each time point). Southern blot analysis was performed on genomic DNA isolated from the liver, lung, and kidney to assess gene transfer of BCRF1. Homogenized liver protein was analyzed for tumor necrosis factor-α (TNF-α), IL-1β, IL-6, and recombinant vIL-10. Results: Southern blot analysis confirmed successful gene transfer to the liver but not to the lung, kidney, or dl434-transduced liver in mice that received adenovectors. Viral IL-10 levels within the liver ranged from 14 to 18 ng/mL. In controls, INF-α production was elevated at early time points, to 18,000 pg/mL, but decreased rapidly by 24 hours after LPS challenge. The TNF-α levels of animals treated with Ad5svBCRF1 were significantly lower than those of controls throughout the course of study ( P < .0001). After the LPS challenge, hepatic IL-1β decreased, from a maximum of 800 pg/mL (2 hours) to 411 pg/mL (24 hours). Inhibition of IL-1β by vIL-10 occurred at 1 hour ( P < .016) and 2 hours ( P < .001) only. Hepatic production of IL-6 after LPS challenge ranged from 7 to 8,000 pg/mL in all groups and was not altered by vIL-10 gene therapy. Conclusion: In vivo administration of adenovectors encoding BCRF1 to newborn mice results in efficient hepatic transduction and expression of recombinant vIL-10. The Kupffer cell response to LPS is suppressed with respect to TNF-α and IL-1β, but not IL-16. In vivo modulation of hepatic cytokine responses is achievable using gene products that mimic cellular cytokines. This is an effective model for the selective evaluation of therapeutic gene products for gene therapy of sepsis.