Christian Clarke

Large-Scale Production of High-Quality Helper-Dependent Adenoviral Vectors Using Adherent Cells in Cell Factories

The most efficient and widely used system for generating helper-dependent adenoviral vectors (HDAds) is the Cre/loxP system developed by Graham and co-workers (Parks, R.J., Chen, L., Anton, M., Sankar, U., Rudnicki, M.A., and Graham, F.L. [ 1996 ]. Proc. Natl. Acad. Sci. U. S. A. 93, 13565-13570). Alternative systems have been developed for HDAd production, but all are limited by the technical complexity of a three-component vector production system for reproducibly generating large quantities of adenovirus with high infectivity and low helper virus (HV) contamination. Recently, these problems were addressed by Ng and co-workers (Palmer, D., and Ng, P. [ 2003 ]. Mol Ther. 8, 846-852), who developed an improved system that combines the use of a suspension-adapted producer cell line expressing high levels of Cre recombinase, a HV resistant to mutation, and a refined purification protocol. With this system, >1 x 10(13) highly infectious vector particles are easily produced without vector genome rearrangements and having very low HV contamination levels. However, the Ng system incorporates a spinner flask culture system that involves considerable time, effort, and tissue culture medium to produce HDAds. We have an alternative system to obtain comparable quantities with equivalent quality to the spinner flask approach but requiring reduced labor and lower volumes of medium. This method utilizes a 10-chamber cell factory with adherent cells to produce high infectivity of HDAds with minimal HV contamination while improving yield and reducing technical complexity, effort, and medium requirements. This system is easily translatable to the production of clinical-grade HDAds for human trials.

MyD88-Dependent Silencing of Transgene Expression During the Innate and Adaptive Immune Response to Helper-Dependent Adenovirus

Activation of the host innate immune response after systemic administration of adenoviral vectors constitutes a principal impediment to successful clinical gene replacement therapies. Although helper-dependent adenoviruses (HDAds) lack all viral functional genes, systemic administration of a high dose of HDAd still elicits a potent innate immune response in host animals. Toll-like receptors (TLRs) are innate receptors that sense microbial products and trigger the maturation of antigen-presenting cells and cytokine production via MyD88-dependent signaling (except TLR3). Here we show that mice lacking MyD88 exhibit a dramatic reduction in proinflammatory cytokines after intravenous injection of a high dose of HDAd, and show significantly reduced induction of the adaptive immune response when compared with wild-type and TLR2-deficient mice. Importantly, MyD88(-/-) mice also show significantly higher and longer sustained transgene expression than do wild-type mice. Chromatin immunoprecipitation studies using wild-type and MyD88-deficient primary mouse embryonic fibroblasts showed significant MyD88-dependent transcriptional silencing of the HDAd-encoded transgenes. Our results demonstrate that MyD88 signaling, activated by systemic delivery of HDAd, initiates an innate immune response that suppresses transgene expression at the transcriptional level before initiation of the adaptive immune response.

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.

627. Calcium Phopspate Precipitation of Adenovirus Enhances Gene Delivery into Human Dendritic Cells

Top of pageAbstract Mature dendritic cells (DC) are potent antigen presenting cells (APC) that have been used in vaccine studies as well as adoptive immunotherapy protocols. Adenovirus (Ad) vectors are frequently used to genetically modify DC; however gene transfer with recombinant adenovirus (serotype 5) into human DC is inefficient since DC do not express the cognate Ad5 receptor. Calcium phosphate (CaPi) precipitation of Ad has been shown to enhance gene transfer into murine DC and the aim of this study was to evaluate if CaPi precipitation enhances the gene delivery into human DC without interfering with their ability to activate antigen-specific T cells. Immature, monocyte derived DC were transduced with Ad5 or Ad5:CaPi complexes and gene expression was determined using GFP as a marker gene. CaPi precipitation of Ad5 increased DC gene transfer on average 35-fold when compared to unmodified Ad5. Ad5:CaPi complexes mediated gene delivery was similar in direct comparison to the optimal Ad for DC gene transfer (Ad5f35). Moreover, CaPi also increased the transduction efficiency of Ad5f35 at least 2-fold. CaPi transduction of immature DC did not reduce cell viability or their ability to be matured as judged by expression of CD83, and DR. To determine if Ad:CaPi treated DC can reactivate antigen-specific T cells, DC were incubated with an Ad5f35 vector encoding the subdominant Epstein Barr virus (EBV) antigens, latent membrane proteins (LMP) 2, which is expressed in EBV-positive malignancies. CaPi precipitation increased the expression of LMP2 in human DC 5|[ndash]|10 fold compared to virus alone, resulting in enhanced activation of LMP2-specific T cells in vitro. In conclusion, CaPi complexes increased gene transfer into human DC with both Ad5 and Ad5f35 vectors without changing their phenotype or their ability to activate antigen-specific T cells. Ad5:CaPi mediated transduction resulted in similar transduction as Ad5f35 vectors, which allows the use of existing Ad5 vector technology to genetically modify human DC.

598. In-Vivo Test of Coagulation Factor VIII with Improved Properties for Hemophilia A Gene Therapy by Helper-Dependent Adenoviral Vectors

Top of pageAbstract Hemophilia A is an X-linked bleeding disorder resulting from a deficiency of coagulation factor VIII (FVIII). FVIII expression is limited by unstable mRNA, interaction with endoplasmic reticulum (ER) chaperones, a requirement for facilitated ER to Golgi transport through interaction with the mannose-binding lectin LMAN1 and the instability of the activated form of FVIII (FVIIIa). Bioengineering strategies, like the development of different variants of recombinant FVIII molecules, by rational design can overcome these limitations. Helper-dependent adenovirus (HDV) can facilitate transfer of up to 36 kb transgene DNA and is optimal for the study of FVIII and its variants. In this study we evaluated the in vivo efficacy of six different variants of recombinant FVIII. To this end, we generated six different helper dependent adenoviral vectors carrying human cDNAs regulated by the liver tissue restricted PEPCK promoter. To compare the short term efficacy and duration of hFVIII expression and infectivity of the different HDV hFVIII variants, FVIII deficient mice were treated with each vector at a dose of 5X1012 vp/kg via tail vein injection. Plasma was collected at baseline, two and four weeks post-injection and FVIII biological activity was quantified by Coatest chromogenic bioassay. In this experiment, all vectors expressed functional FVIII at two weeks post-injection. FVIII activity varied from 2-8% in WT FVIII to up to 70% in some other constructs. Functional FVIII activity dropped significantly in all vectors by 4 weeks except in the mice treated with an inactivation resistant rFVIII (HDV IR8 hFVIII) suggesting that variant may exhibit an improved immunological profile. Interestingly, another construct, containing the F309S substitution, which decreases binding to BiP and thus increases the rate of secretion of the FVIII mutation, appears to significantly improve activity over WT hFVIII in vivo.