124. Efficient Transduction and Activation of Dendritic Cells by Adenoviral Vectors Targeted to DC-SIGN

ADENOVIRUS TARGETING and the risk of liver damage. Therefore, development of CARindependent gene transfer strategy to the lung is needed. We recently constructed an Ad in which the fiber was replaced by human CD40 ligand (Ad5 Luc FF / CD40L) and showed the utility of this fiber replacement strategy for genetic targeting of the virus to human CD40 (Belousova et al. J. Virol 77:11367-77, 2003). In addition, we recently showed the promoter for vascular endothelial growth factor receptor 1 (fms-like tyrosine kinase-1; flt-1) has high activity in pulmonary endothelial cells, low activity in hepatocytes (Nicklin et al. Hypertension 38:65-70, 2001), and also provided transcriptional targeting to the lung vasculature (Reynolds et al. Nat. Biotechnol.19:838-42, 2001). We hypothesized that hCD40 expression, transcriptionally targeted to the vascular endothelium via the flt-1 promoter, could provide CD40-targeted gene delivery to the lung in a CAR-independent manner. We constructed an E1deleted recombinant adenovirus expressing hCD40 driven by the flt-1 promoter (Ad5flt1-hCD40). We then analyzed hCD40 expression and flt-1 activity in several cell lines by luciferase assay and fluorescence-activated cell sorting (FACS) analysis. We selected NTERA-2 cells as low hCD40 and flt-1 positive cell line, and HeLa cells as low hCD40 and flt-1 negative cell line as control for further experiments. Forty-eight hours after the first infection with Adflt1hCD40 and the control virus, we quantitated hCD40 expression on the cell surface by FACS analysis. Adflt1-hCD40 infected NTERA2 cells had markedly increased hCD40 expression compared to either control virus infected cells or flt-1 negative HeLa cells. Further, we evaluated infectivity enhancement gained by increased hCD40 in the flt-1 positive cell line. Forty-eight hours after the first infection, cells were infected with Ad5 Luc FF / CD40L vectors at an MOI of 1000 viral particles / cell to determine infectivity enhancement accrued via CD40 targeting. Luciferase assays were performed 24 hours post second infection. Ad5 Luc FF / CD40L vector infectivity in Ad flt-1 hCD40 infected NTERA-2 cells showed 13-fold greater luciferase reporter gene activities versus negative control virus infected cells. These data suggest that expression of hCD40 in pulmonary endothelial cells using recombinant Ad vectors containing hCD40 gene under the control of the flt-1 promoter and targeted with a novel fiber modified Ad vector can increase the efficacy and specificity of adenoviral gene therapy for lung disease.

123. Adenovirus Type 11 Based Vectors as a Platform for Treatment of Metastatic and Hematopoietic Disease Daniel Stone,1 Andre Lieber.1 Division of Medical Genetics, University of Washington, Seattle, WA.

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Adenovirus (Ad) vectors derived from group C serotype Ad5 have shown great promise for treatment of disease based on their ability to efficiently infect multiple therapeutic target cell populations. Despite this ability these vector systems do not efficiently infect all potential disease target cell types, due to a lack of primary attachment receptors on target cells, and are hampered in vivo by native virus neutralizing immune responses and acute vectormediated toxicity. In an attempt to circumvent these limitations to Ad-mediated gene therapy we have developed a vector system based on group B serotype Ad11 whereby first generation (E1 deleted) vector genomes can be generated by E.coli recombination or by cotransfection of 2 shuttle plasmids into the cell line 293Ad11E1B55K. Vector preparations of > 1012 particles/ml can be generated with the optimum cell harvest time being 72 hours after infection. Competition experiments using recombinant Ad11p fiber knob to block binding of the vector Ad11-CMV-GFP to CHO cells expressing the previously identified adenovirus receptors CAR and CD46 demonstrated that Ad11 vectors are able to infect cells through a CAR-independent fiber knob/CD46 mediated pathway. Analysis S48

of serum samples taken from normal US or normal and cervical cancer positive Senegalese volunteers demonstrated that virus directed neutralizing antibodies (VNAbs) against Ad11 were present in less than 10% of samples whilst VNAbs against Ad5 were present in more than 30% of samples. Transformation assays in baby rat kidney (BRK) cells transfected with wild type or E1 deleted genomes from Ad5 or Ad11 showed no transformed foci in E1 deleted genome transfected cells of either serotype whilst wild type Ad5 showed more foci than wild type Ad11. Transduction studies using Ad5 and Ad11 vectors expressing the reporter gene GFP from the CMV promoter were also carried out. A panel of human tumor cell lines derived from cervix, lung, liver, breast, prostate, colon, ovary, blood and brain demonstrated transduction profiles for Ad5 and Ad11 derived vectors that correlated with levels of CAR and CD46 expression. Primary human cord blood derived CD34+ve cells and peripheral blood derived dendritic cells were readily infectable with Ad11 derived vectors in contrast to vectors based on Ad5. Additionally we previously developed an Ad5 based system (Ad.IR) in which tumor specific replication of E1 deleted Ad DNA upon infection mediates expression of a toxic protein. Replication of Ad11 vector DNA in a panel of tumor cells demonstrated the suitability of Ad11 based vectors for application in an Ad.IR system. In summary we have developed E1 deleted Ad11 vectors that can utilize CD46 for cell infection, have a low prevalence to neutralization by human serum and are non-transforming. These vectors provide an excellent platform for the treatment of diseases in which Ad5 based vectors have shown limitations including treatment of metastatic and hematopoietic disease.

124. Efficient Transduction and Activation of Dendritic Cells by Adenoviral Vectors Targeted to DC-SIGN Nikolay Korokhov,1 Wayne A. Aldrich,3 Alex Krendelchtchikov,1 Angela Granelli-Piperno,5 Papia T. Banerjee,4 Stephen D. Gillies,4 Pierre L. Triozzi,3 Abner M. Mhashilkar,1 David T. Curiel.2 1 Vectorlogics, Inc., Birmingham, AL; 2Division of Human Gene Therapy, Departments of Medicine, Pathology and Surgery and the Gene Therapy Center; 3Division of Hematology-Oncology, Department of Medicine, University of Alabama at Birmingham, Birmingham, AL; 4EMD Lexigen Research Center, Billerica, MA; 5 Laboratory of Cellular Physiology and Immunology, The Rockefeller University, New York, NY. Dendritic cells (DCs) are a central element of the development of antigen-specific immune responses. The lack of a specific and efficient technique for delivery of antigens to DCs remains the major obstacle, limiting the vaccine’s ability to induce an effective immune response. In this regard, adenoviral (Ad) vectors offer a number of significant advantages for this application. Further gains in the utility of Ad would accrue to the ability to alter tropism such that DCselective transduction could be achieved. Immature DCs “monitor” the environment via antigen recognition receptors including the Tolllike receptors and the C-type lectins. The C-type lectin, DC-SIGN, is an internalizing receptor, and, due to high level expression by monocyte-derived DCs is an attractive receptor for targeting Ad vectors for DC-based tumor vaccination approaches. Here, we have shown that Ad vectors targeted to DC-SIGN can efficiently and specifically infect DC-SIGN-positive cells. Our strategy employed a genetically modified Ad vector allowing formation of a protein bridge between a targeting ligand and the modified protein of the viral capsid. The components included (1) an antibody against DC-SIGN or a fused complex of Ig-Fc with ICAM-3, a natural ligand for DC-SIGN and (2) an Ad vector harboring the Fc-binding domain of Staphylococcus aureus Protein A, genetically incorporated in the Ad fiber, which functions as a docking domain for binding with the Fc domain. We have shown that Ad Molecular Therapy Volume 9, Supplement 1, May 2004

Copyright  The American Society of Gene Therapy

ADENOVIRUS TARGETING vectors targeted with antibody to DC-SIGN allow efficient DC transduction. Moreover, transduction of dendritic cells by the DCSIGN-targeted vectors resulted in maturation and activation of the DCs, as evidenced by increased expression of CD83, CD86 and by the production of IL-12. In conclusion, the DC-activating properties together with very efficient delivery of the gene of interest to the DCs via DC-SIGNtargeted vector suggesting this as an effective means for delivery of antigen to dendritic cells for genetic immunization.

in a 4.7-fold increase in gene expression for the composite formulation, having no effect on the free AdV treated cells. Conclusions Immobilization of AdV on a magnetic polymer-based NP platform resulted in a substantial increase in the efficacy of gene transfer in vitro. Rendering this formulation magnetically-responsive provides a potential means for optimizing its biodistribution and efficacy in vivo, and makes this novel gene delivery system an interesting candidate for therapeutic applications.

125. Formulation and In Vitro Properties of Adenovirus-Polylactide Nanoparticle AffinityBased and Magnetic Composites

126. Zc-Ad Vector: A Novel Immunotargeting Vector with a Modified Fc-Binding Domain

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Michael Chorny, Ilia Fishbein, Ivan S. Alferiev, Origene Nyanguile,1 Robert J. Levy.1 1 Department of Cardiology Research, The Children’s Hospital of Philadelphia, Philadelphia, PA. Background Adenoviral vectors have shown promise as a tool for gene deliverybased therapeutic applications. Their use is however limited by the reduced efficacy and systemic adverse reactions resulting from the inability to effectively localize and provide the sustained presence of the vector in the target tissue, while minimizing its escape from the delivery site. To address these problems we investigated the affinity immobilization of adenovirus (AdV) on a biodegradable nanoparticulate platform. Additionally we explored the possibility of enhancing the gene expression by rendering the composite magnetically responsive. Methods Two polylactide-based nanoparticle (NP) formulations were formed by the nanoprecipitation and emulsification-solvent evaporation methods. The NP surface modification with an anionic thiol-reactive derivative of polyallylamine was accomplished photochemically by a brief exposure to long-wave UV light. After separation from unreacted polymer NP were coated with a thiolated form of D1 domain of the Coxsackie-AdV receptor and finally incubated with GFP-encoding AdV suspension in presence of 5% bovine serum albumin. Magnetic NP were formed by inclusion of iron oxide nanocrystals in the polymeric matrix. The particle core and the virus were stained with green BODIPY and red Cy3 dyes, respectively, for fluorimetric studies. AdV-NP binding efficacy was evaluated by measuring the residual fluorescence of unbound virus following magnetic separation of AdV-NP composite. The formulation uptake and efficacy were studied on rat arterial smooth muscle cell culture. The intracellular localization was determined on live cells as a function of incubation time and particle size. The effect of magnetic force on gene expression was assessed by measuring GFP fluorescence in cell lysates using free AdV as a control. Results NP sized 160 and 360 nm were surface-modified with a thiolreactive polymer having a particle stabilizing effect due to its negative charge. The colloidal stability of the NP was not adversely affected by their subsequent modification with D1 protein and AdV. Five and 20 min photoactivation resulted in an equally high NP capacity (∼92%) for AdV binding, whereas unspecifically bound AdV fraction was consistently below 25% in the studied concentration range. Smaller NP exhibited a 1.2-fold higher in vitro cellular uptake and a more rapid elimination than that of larger NP; about 14% of the 360 nm NP initially taken up were found in the cells after 48 hr, while 160 nm NP were undetectable at this timepoint. GFP expression mediated by AdV-particle composite was 2.4 and 10.2-fold higher than that of free AdV when incubated with or without magnet, respectively. The magnetic field exposure resulted Molecular Therapy Volume 9, Supplement 1, May 2004 Copyright  The American Society of Gene Therapy

Sam C. Noureddini,1 Nikolay Korokhov,1 Abner Mhashilkar,1 Joanne T. Douglas,2 David T. Curiel.2 1 VectorLogics, Inc., Birmingham, AL; 2Division of Human Gene Therapy, Department of Medicine, Pathology, and Surgery, and the Gene Therapy Center, University of Alabama at Birmingham, Birmingham, AL. Adenovirus serotype 5 (Ad5) has shown potential as a gene delivery vehicle for numerous gene therapy applications. Because of the paucity of Ad5’s primary receptor, coxsackie-adenovirus receptor (CAR), on cancer cells, and its widespread distribution on other cell types, vector targeting to specific receptors on cancer cells has become a mandate for efficient gene transfer. In one targeting scheme, the receptor-selective affinity of immunoglobin G (IgG) molecules has been employed to retarget Ad5 to specific cell types. This system provides for a flexible, modular approach to targeting cancer cells, because of the vector’s ability to utilize any of the multiple IgG molecules with characterized receptor affinity. In this targeting schema, the Ad5 fiber capsid protein has been engineered to contain a specific domain (Cd) of protein A from S. aureus, exhibiting binding affinity for the conserved Fc domain common to all IgG molecules. Attempts to fully develop this technology, however, have been confounded by high degrees of viral aggregation, observed after viral-IgG complexes have been formed ex vivo, thus hindering the vector’s ability to transfer genes. The proposed cause of this aggregation is the Cd domain’s ability to bind the Fab regions of the IgG molecule, in addition to the Fc domain, yielding complexes of multiple virions crosslinked via IgG molecules. To circumvent this problem, we have engineered a novel IgG binding ligand, the Zc domain; a modification of the previous Fc-binding domain employed for this targeting schema. Based on the literature of well-known non-Fab-binding, Fc-binding domains, we have abolished the Zc domain’s ability to bind the Fab regions of IgG molecules, via sitedirected mutagenesis of a single glycine to alanine substitution. With this structural modification, we hypothesize that the degree of aggregation displayed in Ad-Ig complexes will be lessened, thus, enhancing the vector’s ability to transfer genes in situ. Given the promise shown for modifications of this nature at the C-terminus, we have constructed an E1-deleted Ad5 vector containing a C-terminal linker with the Zc domain, and enhanced green fluorescent protein (GFP) as a reporter gene. The Zc-Ad vectors were propagated and incubated with either a monoclonal anti-CD40 IgG, or an Fc antiCD40 single chain antibody (scFv) fusion protein to provide for CD40 targeted Ad5 vectors. Gene transfer analyses, comparing the targeting efficacy of the Zc vs. Cd viruses, complexed with the IgG and scFv fusion, were carried out with 293 cells expressing CD40. Preliminary data for these experiments demonstrate that the Zc-Ad vector is equally effective as our previous Ig-binding vector system, as displayed by CAR-independent, CD40-mediated gene transfer in vitro. Because aggregation has apparently undermined the potential utility of targeting by Ig-binding Ads hitherto, we propose that the Zc ligand will mitigate aggregation, and optimize these vectors for efficient gene transfer. S49