153. Adenoviral Gene Therapy Vectors Expressing the Adenovirus Death Protein Demonstrate Increased Transgene Expression In Vivo as Demonstrated by Dose-Volume Histogram Analysis of Reporter Gene Activity

ADENOVIRUS VECTORS: GENERAL BIOLOGY AND HOST RESPONSE 151. Evaluation of a Serotype 35 Fiber Containing Adenovirus Vector for Vaccination Nelson C. Di Paolo,1 Shaoheng Ni,1 Anuj Gaggar,1 Zong-Yi Li,1 Andre Lieber.1 1 Medicine, University of Washington, Seattle, WA. Adenovirus (Ad) vectors containing fibers from group B serotypes (such as Ad35) efficiently transduce dendritic cells (DC) in vitro and are considered candidates for vaccination or immunotherapy approaches. However, in vivo, “adjuvant” effects attributed to Ad might be less for Ad5/35 than for Ad5 vectors because injection of an Ad5 vector containing Ad35 fibers (Ad5/35) resulted in lower level of pro-inflammatory cytokines than that seen after injection of unmodified Ad5 vector. Moreover, signaling through the Ad5/35 receptor, CD46, in DC and/or T-cells has been implicated in induction of immunological anergy. We aimed to: i) test, in a CD46 transgenic mouse model and in human peripheral blood mononuclear cells (PBMC), the efficacy of Ad5/35 vectors to mount an antigen specific immune response, and ii) analyze the effect of Ad5/35 injection into CD46 transgenic and wild-type mice on the immune status of the host. Intravenously injected Ad5/35 vectors into CD46 tg mice showed no significant transduction in most organs except the spleen, where we found Ad5/35-mediated transgene expression in the marginal zone of the red pulpa, in CD11c+ cells that most likely represent antigen-presenting cells (APC). This was not seen after infusion of an Ad5 vector. We are evaluating the transduction of APC upon intramuscular and subcutaneous Ad5 and Ad5/35 injection and data will be presented. We also found that plasma levels of TNF-α, IL6, INF-γ and MCP-1 were lower than in mice injected with Ad5 vectors. To characterize whether the transduction of splenic DC affects CD4+ T cell mediated immune responses, we performed an in vivo contact hypersensitivity (CH) assay. Wt and CD46 tg mice were injected with saline, Ad5 or Ad5/35 vectors. We found a slightly but significantly reduced CH response in Ad5/35 injected CD46 tg mice compared to all other groups, which would indicate unresponsiveness in CD4+ T cells. In order to analyze the immune response to a transgene expressed from Ad vectors in this model, we injected CD46 tg mice intramuscularly with Ad5 or Ad5/35 vectors expressing a model hepatitis B virus antigen, HBeAg. Fourteen days later, we analyzed by ELISpot assay the frequency of INF-γ secreting T cells to both HBeAg and the vector. There was no significant difference in the frequency of HBeAg T-cells between groups, indicating that the use of Ad5/35 vectors do not result in a stronger vaccination effect. We also observed a high frequency of Ad-specific T-cells, which suggests that CD46 signaling did not inhibit an anti Ad T-cell response. Finally, we analyzed by ELISpot assay, in an in vitro model with human cells, the frequency of INFγ secreting T cells specific to an antigen and to Ad vectors. Human DC from a CMV positive donor were mock transduced or with Ad5 or Ad5/35 vectors, pulsed (or left untreated) with an immunogenic CMV peptide, pp65, and incubated with autologous PBMC. We found that CD46 signaling did not modify the immune response to the CMV peptide or the Ad. Conclusion: we did not find evidence that Ad5/35 vectors induce a stronger antigen specific immune response than an Ad5 vector, nor did we see convincing evidence that Ad5/35 vectors modified the anti Ad immune response.

Molecular Therapy Volume 11, Supplement 1, May 2005 Copyright  The American Society of Gene Therapy

152. Coxsackievirus-Adenovirus Receptor (CAR) and αvβ3 or αvβ5 Integrin Independent Internalization of Porcine Adenoviral Vectors: Implications in Gene Therapy Dinesh S. Bangari,1 Suresh K. Mittal.1 1 Pathobiology, Purdue University, West Lafayette, IN. Nonhuman adenoviruses including porcine adenovirus serotype 3 (PAd3) are emerging vectors for gene delivery. PAd3 efficiently transduces human and murine cells and circumvents preexisting humoral immunity in humans. Coxsackievirus and adenovirus receptor (CAR) is the primary receptor and αvβ3 or αvβ5 integrin is involved as a secondary receptor for various human adenovirus (HAd) subtypes including HAd5. In this study we deduced the role of CAR, αvβ3 or αvβ5 integrin in PAd3 internalization. Transduction experiments were conducted in human mammary epithelial (MCF10A) cells using replication defective PAd-GFP (PAd3 vector expressing green fluorescent protein [GFP]) and HAd-GFP (HAd5 vector expressing GFP). MCF-10A cells were treated with or without anti-human CAR, or anti-αvβ3 or anti-αvβ5 integrin antibodies prior to infection with HAd-GFP or PAd-GFP. Significant (P<0.05) inhibition in transduction by HAd-GFP was observed in the antibody treated cells as compared to the untreated cells, whereas transduction by PAd-GFP remained to similar levels irrespective of the treatment. To study fiber knob-mediated virus interference, MCF-10A cells were treated with or without the recombinant HAd5 or PAd3 knob followed by infection with HAd-GFP or PAd-GFP. Significant (P<0.05) inhibition was observed only in transduction of the homologous vector. These results suggested that PAd3 internalization was CAR- as well as αvβ3 or αvβ5 integrin-independent and the primary receptor for HAd5 and PAd3 were distinct. CAR- and αvβ3 or αvβ5 integrin-independent entry of PAd3 vectors may have implications in targeting cell types that are not efficiently transduced by other adenoviral vectors.

153. Adenoviral Gene Therapy Vectors Expressing the Adenovirus Death Protein Demonstrate Increased Transgene Expression In Vivo as Demonstrated by Dose-Volume Histogram Analysis of Reporter Gene Activity Kenneth N. Barton,1 Stephen L. Brown,1 Jae Ho Kim,1 Hans Stricker,2 Kastytis Karvelis,3 Mei Lu,4 Svend O. Freytag.1 1 Radiation Oncology, Henry Ford Health System, Detroit, MI; 2 Urology, Henry Ford Health System, Detroit, MI; 3Radiology, Henry Ford Health System, Detroit, MI; 4Biostatistics & Epidemiology, Henry Ford Health System, Detroit, MI. A current limitation of cancer gene therapy is the lack of significant efficacy in the clinic. To improve the effectiveness of adenovirusdelivered gene therapy for the treatment of cancer, we have evaluated various methods to increase transgene expression in vivo. One such method was to co-express the adenoviral death protein (ADP) in a replication-competent adenovirus (Ad5-hNISrep-ADP) also expressing the human sodium iodide symporter (hNIS) as a reporter gene. The ADP gene product has been shown by others to increase cell lysis and adenovirus spread in vitro. The hNIS gene product enables adenovirus-infected cells to uptake the radioactive tracer, 99m pertechnetate (99mTcO4-), which can be imaged non-invasively using gamma camera scintigraphy, and the volume of transgene expression can be quantified in thin tissue sections using dose-volume histogram (DVH) analysis. To study the possible effect of ADP on transgene expression in vivo, the volume of transgene expression was compared following direct injection of replication-competent adenoviruses lacking (Ad5-hNISrep) or containing (Ad5-hNISrepADP) ADP into contralateral lobes of the naïve canine prostate and S61

“OTHER” DNA VIRUS VECTORS into human tumor xenografts implanted bilaterally in the hind legs of Balb/c mice. Following injection of 1 x 1011 vp (0.1 ml) in the canine prostate, the volume of hNIS-dependent uptake of 99mTcO4with Ad5-hNISrep-ADP was on average 3-fold greater than with Ad5-hNISrep (5.0% ± 1.3% vs. 1.5% ± 0.5% of the prostate volume, respectively; n = 3). Similarly, following injection of 1 x 1010 vp (0.05 ml) in human tumor xenografts (150 mm3), the extent of hNIS activity as a percentage of the tumor volume was 16.2% ± 9.5% for Ad5-hNISrep-ADP versus 5.8% ± 2.5% for Ad5-hNISrep (n = 6), representing a 2.3-fold increase in transgene expression when ADP was present. These results indicate that inclusion of ADP in adenoviral gene therapy vectors can significantly improve the volume of transgene expression in vivo, which may translate into improved therapeutic efficacy when used in a clinical setting.

154. Relationship between the Expression of β3 and Membrane Receptors (CAR, Integrin αvβ β5 ) and Adenoviral Transduction Integrin αvβ Efficiency Hiroyuki Inoue,1 Terumasa Hisano,1 Yukoh Nakazaki,1 Gaku Sakaguchi,1 Ryo Kurita,1 Koichi Takayama,2 Yoichi Nakanishi,2 Kenzaburo Tani.1 1 Department of Clinical Genetics, Medical Institute of Bioregulation,Kyushu University, Fukuoka, Japan; 2Research Institute fo Diseases of the Chest, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan. Recent reports demonstrated that GM-CSF (Granulocyte Macrophage colony-Stimulating Factor)-transduced tumor vaccines (GVAX) had substantial antitumor activity in preclinical cancer models and partially in clinical situations because of their chemotactical attraction and activation of dendritic cells. To produce GVAX efficiently, recombinant type 5 E1 deleted adenovirus encoding human GM-CSF is one of the most promising vectors (AdenoGVAX). Adenoviral gene transduction is currently considered to be mediated by CAR (Coxsackie-Adenovirus Receptor), integrinαvβ3 and integrinαvβ5. Although previous reports showed that CAR and the integrin groups could alternatively be used for adenoviral transduction in limited cells, systematical data focusing on the relationship between the expression of membrane receptors (CAR, integrin αvβ3 and integrin αvβ5) and adenoviral transduction efficiency has not been reported. As high GM-CSF production is considered to be required to obtain clinical effects, prediction of GM-CSF production from AdenoGVAX transduced tumor cells is critical to perform the clinical trial efficiently. In this study, we reported the relationship between the expression of membrane receptors and adenoviral mediated GM-CSF production rate in renal cell carcinoma (RCC) cells and non small lung carcinoma (NSCLC) cells. At first, we investigated the expression levels of CAR, integrinαvβ3 and integrinαvβ5 in human 11 NSCLC cell lines, 6 RCC cell lines and 3 RCC primary cells by immunocytochemical staining and FACS analysis. Secondly, we quantified the membrane proteins expression by Western blot analysis. Thirdly, we measured human GM-CSF production from Adeno-GVAX transduced carcinoma cells. In NSCLC cell lines, most of them are CAR positive, but integrin αvβ3 and integrin αvβ5 negative. In contrast, most of the RCC cells were integrin αvβ3 and integrin αvβ5 positive and CAR negative. Interestingly, three RCC cell lines, which produced higher levels of GM-CSF, were positive for all of the three receptors. The GM-CSF production rates in NSCLC cells, however, were heterogenous.

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In conclusion, to obtain the higher GM-CSF production, the expression of both CAR and integrins was considered to be necessary in RCC cells. In NSCLC, the introduction of another molecules are may be required to predict higher GM-CSF production after AdenoGVAX transduction.

“OTHER” DNA VIRUS VECTORS 155. Oral Delivery of Recombinant Vaccinia Viruses Expressing Adjuvanted Islet Autoantigens Protects NOD Mice from Autoimmune Diabetes William H. R. Langridge,1 Bela Denes,1 Nadia Fodor,1 Valentina Krausova,1 Tatyana Timiryasova,1 David Henderson,2 John Hough,2 Jie Yu,1 Istvan Fodor.1 1 Center for Molecular Biology and Gene Therapy, Loma Linda University, Loma Linda, CA.; 2Department of Anatomy, Loma Linda University, Loma Linda, CA. Oral administration of autoantigens can delay or suppress clinical disease in experimental autoimmune disorders. However, repeated feeding of large tolerogen amounts is required over long periods and is only partially effective in animals systemically sensitized to the ingested antigen. The cholera toxin B subunit adjuvant (CTB) when linked to autoantigen epitopes acts as a strong adjuvant to enhance suppression of inflammatory Th1 cell reactivity in both naive and immune animals. Enhanced suppression of inflammatory Th1 cell reactivity was demonstrated in both naive and immune NOD mice following oral inoculation with islet autoantigens coupled to CTB. To stimulate adjuvant-autoantigen fusion protein biosynthesis in the gut mucosae, we evaluated oral inoculation of juvenile NOD mice with recombinant vaccinia virus (rVV) expressing fusion genes encoding CTB linked to the pancreatic islet autoantigens proinsulin (INS) and a 55 kDa peptide from glutamate decarboxylase (GAD55). Oral inoculation with rVV significantly reduced pancreatic insulitis. Hyperglycemia in NOD mice inoculated with CTB::GAD fusion protein was reduced by 40% in comparison with rVV-GAD inoculated mice. Measurement of antibody isotype titers in rVV infected mice suggested activation of autoantigen specific Th2 lymphocytes. These experiments demonstrate the feasibility of vaccinia virus mediated delivery of adjuvanted autoantigens to the mucosae of prediabetic mice for suppression of autoimmune diabetes.

156. Update on Genomic HSV Vector-Related Cytotoxicity: A Vector Preserving the Differentiation Potential of Embryonic and Adult Adipose Stem Cells Julie Fradette,1 Steven K. Wendell,1 Jim Wechuck,1 David M. Krisky,1 Darren Wolfe,1 William F. Goins,1 Joseph C. Glorioso.1 1 Molecular Genetics and Biochemistry, University of Pittsburgh, Pittsburgh, PA. Replication-deficient genomic HSV vectors have been used for transfer of genes to a variety of cell types including neuronal cells, embryonic stem cells as well as adult precursors from human adipose tissue. Human adipocytes and their precursors (Zen-Bio Inc.) are both excellent targets for HSV-mediated transduction and transgene expression (Fradette et al. 2005). Similarly, murine embryonic stem cells express HSV entry receptors, leading to more than 90% of transduced cells at low multiplicity. However, highly defective HSV vectors (e.g. QOZHG) deleted for the majority of immediate early genes including ICP4, ICP27 and ICP22 remain cytotoxic in nonneuronal cells, resulting in the majority of precursor cells dying within 4-5 days post-infection. Therefore, when induced towards differentiation, QOZHG-infected embryonic stem cells do not efficiently generate embryoid bodies (<35%), while preadipocytes Molecular Therapy Volume 11, Supplement 1, May 2005 Copyright  The American Society of Gene Therapy