Viraj P. Mane

Modulation of TNFα, a determinant of acute toxicity associated with systemic delivery of first-generation and helper-dependent adenoviral vectors

Understanding the determinants of the host innate immune response to systemic administration of adenoviral (Ad) vectors is critical for clinical gene therapy. Acute toxicity occurs within minutes to hours after vector administration and is characterized by activation of innate immune responses. Our data indicate that in mice, indicators of vector toxicity include elevations of cytokine levels, liver transaminase levels and thrombocytopenia. To discern potential targets for blunting this host response, we evaluated genetic factors in the host response to systemically administered first-generation Ad vectors (FGV) and helper-dependent Ad vectors (HDV) containing β-galactosidase expression cassettes. A preliminary screen for modulation of vector-induced thrombocytopenia revealed no role for interferon-γ, mast cells or perforin. However, vector-induced thrombocytopenia and interleukin 6 (IL-6) expression are less evident in tumor necrosis factor alpha (TNFα)-deficient mice. Moreover, we also demonstrated that TNFα blockade via antibody or huTNFR:Fc pretreatment attenuates both thrombocytopenia (>40% increase in platelet count) and IL-6 expression (>80% reduction) without affecting interleukin 12 , liver enzymes, hematological indices or vector transduction in a murine model. Our data indicate that the use of HDV, in combination with clinically approved TNFα immunomodulation, may represent an approach for improving the therapeutic index of Ad gene therapy for human clinical trials.

Phenotypic Correction of Ornithine Transcarbamylase Deficiency Using Low Dose Helper Dependent Adenoviral Vectors

Helper‐dependent adenoviral vectors (HDAd) can mediate long‐term phenotypic correction in the ornithine transacarbamylase (OTC)‐deficient mice model with negligible chronic toxicity. However, the high doses required for metabolic correction will result in systemic inflammatory response syndrome in humans. This acute toxicity represents the major obstacle for clinical applications of HDAd vectors for the treatment of OTC deficiency. Strategies for reducing the dose necessary for disease correction are highly desirable because HDAd acute toxicity is clearly dose‐dependent.

535. Long-Term Correction of Hyperbilirubinemia in a Rat Model of Crigler-Najjar Syndrome Type 1

Top of pageAbstract Crigler-Najjar syndrome Type 1 is a recessively inherited disorder caused by a deficiency of the hepatic enzyme bilirubin uridine diphosphate-glucuronosyltransferase (UGT1A1), and is manifested as severe unconjugated hyperbilirubinemia. Unconjugated, water-insoluble bilirubin is not excreted and therefore accumulates in blood and in the central nervous system. This accumulation is fatal to a newborn if untreated. Phototherapy is currently utilized, owing to the ability of blue light to break bilirubin down into water-soluble fragments. However, its efficacy is reduced upon thickening of the skin and further reduced as a patients surfavolume ratio changes around puberty. The alternative, liver transplantation, is neither easily affordable nor widely accessible. We have evaluated an adenoviral gene therapy modality for restoration of UGT1A1 function, over the long term, to the Gunn rat model of Crigler-Najjar Syndrome. We employed a helper-dependent adenoviral vector, whose lack of viral coding sequences allows greater persistence of transgene expression coupled to reduced chronic toxicity, as compared to early-generation adenoviral vectors. The vector contains the human UGT1A1 cDNA driven by the liver-restricted rat PEPCK promoter and a Woodchuck Posttranscriptional Regulatory Element (WPRE). Adenoviral vector was administered to Gunn rats via the tail vein at the following doses: 6, 30 and 100 (|[times]|1011 viral particles/kilogram). Treatment did not cause significant elevation of serum transaminases which are indicative of hepatocyte injury. Furthermore, moderate thrombocytopenia developed after vector administration but had resolved within a few days of injection. A clinically significant reduction in bilirubin levels was observed using the lowest adenoviral dose; this correction was maintained past 67 weeks. In rats receiving intermediate or high doses, complete correction of hyperbilirubinemia was sustained for over 2 years. HPLC analysis of serum processed from terminal bleeds demonstrated a dose-dependent reduction of unconjugated bilirubin, while bile HPLC analysis showed an increase in conjugated (glucuronidated) bilirubin. For an additional in vivo assay of UGT1A1 activity, HPLC analysis was carried out on bile samples taken before and after tail vein injection of bilirubin isomers BR-III and BR-XIII. Conjugation of these isomers requires UGT1A1 activity, and HPLC analysis showed an excess of bilirubin conjugating capacity in rats receiving intermediate or high doses. Taken together, these results support the efficacy of a single systemic administration of a helper-dependent adenoviral vector towards restoring UGT1A1 enzyme function over the long term. Acute toxicities occurred but were quickly resolved. The relatively favorable dose response makes this condition a candidate for considering clinical trials in the future.

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.