Injection Of Helper-dependent Adenoviral Vector Research Articles

Improved Efficacy and Reduced Toxicity by Ultrasound-Guided Intrahepatic Injections of Helper-Dependent Adenoviral Vector in Gunn Rats

Crigler-Najjar syndrome type I is caused by mutations of the uridine diphospho-glucuronosyl transferase 1A1 (UGT1A1) gene resulting in life-threatening increase of serum bilirubin. Life-long correction of hyperbilirubinemia was previously shown with intravenous injection of high doses of a helper-dependent adenoviral (HDAd) vector expressing UGT1A1 in the Gunn rat, the animal model of Crigler-Najjar syndrome. However, such high vector doses can activate an acute and potentially lethal inflammatory response with elevated serum interleukin-6 (IL-6). To overcome this obstacle, we investigated safety and efficacy of direct injections of low HDAd doses delivered directly into the liver parenchyma of Gunn rats. Direct hepatic injections performed by either laparotomy or ultrasound-guided percutaneous injections were compared with the same doses given by intravenous injections. A greater reduction of hyperbilirubinemia and increased conjugated bilirubin in bile were achieved with 1 × 10(11) vp/kg by direct liver injections compared with intravenous injections. In sharp contrast to intravenous injections, direct hepatic injections neither raised serum IL-6 nor resulted in thrombocytopenia. In conclusion, ultrasound-guided percutaneous injection of HDAd vectors into liver parenchyma resulted in improved hepatocyte transduction and reduced toxicity compared with systemic injections and is clinically attractive for liver-directed gene therapy of Crigler-Najjar syndrome.

The transient receptor potential vanilloid 4 ion channel regulates the biosynthetic response of chondrocytes to dynamic loading

The transient receptor potential vanilloid 4 ion channel regulates the biosynthetic response of chondrocytes to dynamic loading

Proteoglycan 4 expression protects against the development of osteoarthritis

Osteoarthritis (OA) is a common degenerative condition that afflicts more than 70% of the population between 55 and 77 years of age. Although its prevalence is rising globally with aging of the population, current therapy is limited to symptomatic relief and, in severe cases, joint replacement surgery. We report that intra-articular expression of proteoglycan 4 (Prg4) in mice protects against development of OA. Long-term Prg4 expression under the type II collagen promoter (Col2a1) does not adversely affect skeletal development but protects from developing signs of age-related OA. The protective effect is also shown in a model of posttraumatic OA created by cruciate ligament transection. Moreover, intra-articular injection of helper-dependent adenoviral vector expressing Prg4 protected against the development of posttraumatic OA when administered either before or after injury. Gene expression profiling of mouse articular cartilage and in vitro cell studies show that Prg4 expression inhibits the transcriptional programs that promote cartilage catabolism and hypertrophy through the up-regulation of hypoxia-inducible factor 3α. Analyses of available human OA data sets are consistent with the predictions of this model. Hence, our data provide insight into the mechanisms for OA development and offer a potential chondroprotective approach to its treatment.

Abstract 5067: Interleukin-7 is an Active Participant in Atherogenesis by Promoting Monocyte/macrophage Arrest on Endothelial Cells

Atherosclerosis is a chronic inflammatory disease, but can regress. Despite the importance of atherosclerotic lesion regression, its mechanisms are not well understood. We induced regression of advanced atherosclerosis in Apoe −/− mice and studied potential mechanisms. A single i.v. injection of helper-dependent adenoviral vector expressing human apoE3 (HDAd-gE3) into Apoe −/− mice resulted in a 28±4% reduction of the advanced atherosclerosis (n= 7 vs. baseline n=8, p=0.012) after 41 weeks. Using this system, we analyzed gene expression profiles in the aorta at day 10, when plasma cholesterol was normalized. Pathway and ontology analyses revealed multiple signaling pathways influenced by the treatment; however, after correction for multiple testing we unexpectedly found that interleukin-7 (IL-7) was significantly downregulated. IL-7 is a nonredundant cytokine for B- and T-cell development. Although several studies have suggested that IL-7 is associated with cardiovascular disease, IL-7 has not been implicated as having a direct role in atherogenesis. IL-7 positive areas in the aorta were also reduced in the HDAd-gE3 group by 53% (n=5– 6/group, p<0.01) after 41 weeks of treatment. IL-7 could be a marker for lesion regression or reduced inflammation, or be directly involved in atherosclerosis. To test this hypothesis, we analyzed the effects of IL-7 on expression of adhesion molecules and chemokines in human aortic endothelial cells (HAECs). IL-7 upregulated adhesion molecules and chemokines which include VCAM-1, ICAM-1, E-selectin and monocyte chemotactic protein-1. This upregulation was time- and dose-dependent and promoted monocyte adhesion to HAECs. Moreover, the increase of plasma IL-7 levels in Apoe −/− mice by HDAd-IL-7 enhanced homing of monocyte/macrophage to the lesions by 2-fold (n=6/group, p<0.01) without increasing plasma cholesterol. In contrast, transplantation of bone marrow cells isolated from Il-7 −/− mice into Ldlr −/− mice reduced macrophage positive areas by 26% after 20 weeks compared with those isolated from wild type mice (n=6/group, p<0.05). These results suggest that IL-7 plays a direct role in atherosclerosis by promoting monocyte/macrophage homing to atherosclerotic lesions.

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.

Toll-like Receptor 9 Triggers an Innate Immune Response to Helper-dependent Adenoviral Vectors

A major obstacle to the clinical application of systemic adenoviral gene replacement therapy is the host innate immune response. Although recent studies have attempted to characterize the cellular basis for this response to systemically administered helper-dependent adenoviral vector (HD-Ad), the underlying molecular components of the innate immune repertoire required to recognize the viral vector have yet to be identified. Here, we show that primary macrophages can sense HD-Ad vectors via the Toll-like Receptor 9 (TLR9) and respond by increasing pro-inflammatory cytokine secretion. Moreover, TLR9 sensing is involved in the rapid innate immune response to HD-Ad in vivo. TLR9 deficiency attenuates the innate immune response to HD-Ad, whereas TLR9 blockade reduces the acute inflammatory response after intravenous injection of the vector. Moreover, HD-Ad upregulates TLR9 gene expression independent of TLR9 function, suggesting that additional innate signaling pathways work cooperatively with TLR9. The identification of the components of the innate immune response to adenovirus will facilitate the development of combinatorial therapy directed at increasing the maximal tolerated dose of systemically delivered adenoviral vectors.

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.

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.

Lifelong elimination of hyperbilirubinemia in the Gunn rat with a single injection of helper-dependent adenoviral vector

Crigler-Najjar syndrome is a recessively inherited disorder characterized by severe unconjugated hyperbilirubinemia caused by a deficiency of uridine diphospho-glucuronosyl transferase 1A1. Current therapy relies on phototherapy to prevent kernicterus, but liver transplantation presently is the only permanent cure. Gene therapy is a potential alternative, and recent work has shown that helper-dependent adenoviral (HD-Ad) vectors, devoid of all viral coding sequences, induce prolonged transgene expression and exhibit significantly less chronic toxicity than early-generation Ad vectors. We used a HD-Ad vector to achieve liver-restricted expression of human uridine diphospho-glucuronosyl transferase 1A1 in the Gunn rat, a model of the human disorder. Total plasma bilirubin levels were reduced from >5.0 mg/dl to <<1.4 mg/dl for >2 yr after a single i.v. administration of vector expressing the therapeutic transgene at a dose of 3 x 10(12) viral particles per kg. HPLC analysis of bile from treated rats showed the presence of bilirubin glucuronides at normal WT levels >2 yr after one injection of vector, and i.v. injection of bilirubins IIIalpha and XIIIalpha in the same animals revealed excess bilirubin-conjugating capacity. There was no significant elevation of liver enzymes (alanine aminotransferase) and only transient, moderate thrombocytopenia after injection of the vector. A clinically significant reduction in serum bilirubin was observed with a dose as low as 6 x 10(11) viral particles per kg. We conclude that complete, long-term correction of hyperbilirubinemia in the Gunn rat model of Crigler-Najjar syndrome can be achieved with one injection of HD-Ad vector and negligible chronic toxicity.

Acute toxicity after high-dose systemic injection of helper-dependent adenoviral vectors into nonhuman primates.

Systemic intravascular delivery of adenoviral (Ad) vectors for liver-directed gene therapy has been widely employed because of its simplicity, noninvasiveness, and potential for high transduction. For first-generation Ad vectors (FGAd), this results in high but transient levels of transgene expression and long-term hepatotoxicity due to viral gene expression from the vector backbone. Furthermore, high doses also result in an acute innate inflammatory response with potentially lethal consequences. Unlike FGAd, helper-dependent Ad vectors (HDAd) contain no viral genes and can provide sustained, high-level transgene expression with negligible long-term toxicity. However, whether the absence of viral gene expression leads to any decrease of acute toxicity in nonhuman primates has yet to be determined. To address this, we injected one baboon with 5.6 x 10(12) HDAd viral particles (VP)/kg and a second with 1.1 x 10(13) VP/kg. Approximately 50% hepatocyte transduction, accompanied by mild and transient acute toxicity, was observed in the animal receiving the lower dose. In the animal receiving the higher dose, 100% hepatocyte transduction, accompanied by lethal acute toxicity, was observed. These results indicate that systemic delivery of HDAd, like FGAd, results in acute toxicity in baboons consistent with activation of the innate inflammatory response, the severity of which is dose dependent, and confirm the hypothesis that Ad-mediated acute toxicity is independent of viral gene expression.

Lifetime correction of genetic deficiency in mice with a single injection of helper-dependent adenoviral vector.

Ideally, somatic gene therapy should result in lifetime reversal of genetic deficiencies. However, to date, phenotypic correction of monogenic hyperlipidemia in mouse models by in vivo gene therapy has been short-lived and associated with substantial toxicity. We have developed a helper-dependent adenoviral vector (HD-Ad) containing the apolipoprotein (apo) E gene. A single i.v. injection of this vector completely and stably corrected the hypercholesterolemia in apoE-deficient mice, an effect that lasted the natural lifespan of the mice. At 2.5 years, control aorta was covered 100% by atherosclerotic lesion, whereas aorta of treated mice was essentially lesion-free. There was negligible toxicity associated with the treatment. We also developed a method for repeated HD-Ad vector administration that could be applied to organisms, e.g., humans, with life spans longer than 2-3 years. These studies indicate that HD-Ad is a promising system for liver-directed gene therapy of metabolic diseases.