20. Construction and Characterization of Adenovirus Vectors Expressing Optimized Blood Stage Antigens of Plasmodium falciparum

ADENOVIRUS VECTORS: HYBRID VECTORS/NOVEL SEROTYPES/TARGETING 18. Comparison of Antitumor Activity of Cytosine Deaminase::Uracil Phosphoribosyl Transferase (CD::UPRT) and Purine Nucleoside Phosphorylase (PNP) Suicide Genes Using Replicative but Non-Disseminative Adenovirus Vectors Jairo Jaime,1 Nadine Jabbour,1 William Parker,2 Eric Sorscher,3 Josephine Nalbantoglu,4 Bernard Massie.1,5,6 1 Groupe de Vecteurs de Génomique et Thérapie Génique, Biotechnology Research Institute, NRC, Montreal, Canada; 2 Southern Research Institute, Birmingham, AL; 3Departement of Medicine, University of Alabama, Birmingham, AL; 4Montreal Neurological Institute, McGill University, Montreal, Canada; 5 INRS-IAF, UQAM, Montreal, Canada; 6Faculté de Médecine, Université de Montreal, Montreal, Canada. Cancer gene therapy using suicide genes has shown great potential in tumor models. To improve efficacy, recent attention has focused on the use of oncolytic vectors. To date this strategy has not resulted in vectors with anti-tumor activities sufficiently potent to sustain complete tumor regression. In this study, we pursued a modified approach that makes use of viral DNA replication but not of oncolysis. This approach is based on the protease-deleted adenovirus vector (AdV) (Oualikene et al. 2000). Such vectors are designed to convert transduced cells into mega-factories for suicide genes. To evaluate this strategy, we compared the activity in vitro of two suicide genes: CD::UPRT and PNP in the Replicative but NonDisseminative (RND) AdVs. In AdVs the CD::UPRT or PNP transgene and the E1A gene were introduced as a bicistronic cassette under the control of the CMV5 promoter. Adenoviruses were generated by introducing the expression cassette in the E1 region of an AdV backbone deleted for E1, E3, and the protease gene. Due to the protease deletion, these AdVs can no longer form viral particles, even following replication resulting from E1A expression. The resulting Ad(dPS)C::U-IRES-E1A, and Ad(dPS)PNP-IRES-E1A were grown in protease-complementing 293 cells. When tested for anti-tumor activities in two human and one murine glioblastoma cell lines and one ovarian cancer cell line, Ad(dPS)PNP-IRES-E1A outperformed its counterpart, the Ad(dPS)C::U-IRES-E1A, by up to 16-fold in human glioblastoma and 5 fold in human ovarian cancer cells. Both RND virus performed better than non replicative counterparts. In bystander assays, the number of transduced cells sufficient to convey the anti-tumor activity to the entire cell population could be reduced by more than 10-and 8-fold when comparing Ad(dPS)PNP-IRES-E1A to Ad(dPS)C::U-IRES-E1A on glioblastoma and ovarian cancer cells respectively. In a threedimensional spheroid culture model, when both RND AdVs were compared, anti-tumor activity was significantly better (ANOVA p<0.001) for Ad(dPS)PNP-IRES-E1A in all cancer cell models, ranging from 3-fold in ovarian cancer up to 10-fold in glioblastoma spheroids. In conclusion, the RND AdV platform substantially improves anti-tumor activity of suicide gene therapy. Using this strategy, the suicide gene PNP performed better than CD::UPRT in both glioblastoma and ovarian cancer cells. This work demonstrates that the protease-deleted AdV platform can provide vectors that are potentially safer than conditional oncolytic AdVs and yet generate sufficient anti-tumor activity to eradicate large tumors. Results with Ad(dPS)PNP-IRES-E1A are promising and work with animal models is in progress.

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19. A New, Plasmid-Based Adenoviral Vectoring System Derived from the Highly Immunogenic, Human Ad4 Strain Zachary C. Hartman,1 Todd Mendelson,1 Andrea Amalfitano.2 Duke University, Department of Pediatrics, Division of Medical Genetics, Durham, NC; 2Department of Microbiology and Molecular Genetics and Pediatrics, Michigan State University, Lansing, MI. 1

Human Adenovirus type 4 (HAd-4) is a human member of the Group E Ads, a group also including simian Ads, the latter of which have been reported to be more immunogenic in vitro. More importantly, significant morbidity associated with natural HAd4 infections has been of major concern to both civilians and the US military, prompting deployment of a live, orally administered HAd4 vaccine. These features suggest that HAd4 is intrinsically more immunogenic than other HAds. To exploit these properties, we report the successful development of a HAd4 plasmid-based vector production system. This system allows maximal flexibility for construction of several classes of replication incompetent (or competent) HAd4 based vectors. Transgene insertion into various regions of the HAd4 genome is facilitated by development of several HAd4 targeted shuttle plasmids. HAD4 based vectors grew well in human 293 cells, and by design have a zero probability of homologous recombination with the E1 genes present in 293 cells. A prototype [E1-,E3-] LacZ Ad4 vector showed significantly higher transduction efficiencies in A549, HeLa, and Hep3B cells, but similar transduction efficiencies in 293, Hepa1-6, and Hep2G cell lines, (relative to an identical Ad5-based vector) thus indicating an expanded vector tropism in vitro. In vivo, de-targeting of the liver despite high dose administrations (5x10E10 HAd4 particles/mouse) was noted, and may be due to the shorter shaft, (and lack of HPSG motifs) on the HAd4 fiber. HAd4-based vectors induced the rapid generation of significantly higher plasma levels of many pro-inflammatory cyto/ chemokines, including IL-6, G-CSF, MIP-1a, RANTES, and MCP1, relative to identical treatments with an HAd5 vector. In summary, the immunogenic properties and altered tropism of HAd4 based vectors, (combined with its successful history of being a safe, enterically deliverable vaccine) makes it potentially very useful in pathogenic and cancer vaccination strategies.

20. Construction and Characterization of Adenovirus Vectors Expressing Optimized Blood Stage Antigens of Plasmodium falciparum Ping Chen,1 Svetlana Konovalova,1 Keith Limbach,2 Maureen E. Stefaniak,2 Noelle B. Patterson,2 Joseph J. Campo,2 Sheng Li,3 Richter King,1 Denise L. Doolan,2 Joseph T. Bruder.1 1 Genvec, Inc, 65 West Watkins Mill Road, Gaithersburg, MD; 2 Naval Medical Research Center, 503 Robert Grant Avenue (Room 3W41/3W16), Silver Spring, MD; 3Malaria Vaccine Initiative, PATH, 7500 Old Georgetown Road, 12th Floor, Bethesda, MD. Malaria is the most devastating parasitic disease affecting humans. Each year there are 300-500 million new infections and 1-3 million deaths, primarily of children in sub-Saharan Africa. The feasibility of a malaria vaccine is supported by the demonstration of protective immunity following exposure to the intact Plasmodium parasite and the decrease in incidence, prevalence, and density of infection with age and exposure. In the latter, the protective immune mechanism is thought to be mediated primarily by antibodies directed against antigens expressed during the blood-stage. Immunization with subunit vaccines incorporating blood stage antigens has the potential to induce protective antibody responses. Adenovectors offer great

Molecular Therapy Volume 13, Supplement 1, May 2006 Copyright  The American Society of Gene Therapy

CARDIOVASCULAR GENE THERAPY potential for the next generation of molecular vaccines. They induce strong and protective immune responses in multiple disease systems and multiple animal models including mice and non-human primates. Moreover, adenovectors are now undergoing clinical testing for application as an HIV vaccine. Our strategy is to develop a bivalent adenovector that expresses optimized forms of two P. falciparum blood stage antigens, PfAMA1 and PfMSP142. To maximize the potential of these antigens to induce strong antibody responses, we have designed adenovectors to express these antigens either intracellularly or at the cell surface. In addition, as the malaria parasite does not glycosylate its proteins efficiently, we generated mutants of both PfAMA1 and PfMSP142 with conservative substitutions in all of the potential glycosylation sites. These variant antigens were then built into adenovectors and evaluated for cell surface expression, glycosylation and their capacity to induce antibody and T-cell responses in mice. The higher apparent molecular weight of the secreted/glycosylated (SG) versions of both antigens observed on immunoblots suggested that these antigens were post-translationally modified. The glycosylation status of both antigens was confirmed by treatment with endoglycosidases Endo H and PNGase F. Immunofluorescence assays (IFA) indicated that the PfAMA1 (SG) antigen was located at the cell surface and the secreted/non-glycosylated (SNG) and non-secreted (NS) versions were expressed preferentially inside the cell. Protease digestion of intact infected cells confirmed these findings, suggesting that the PfAMA1 (SG) antigen is present at the cell surface in a conformation that is recognized by the 4G2 antibody and sensitive to trypsin digestion. All variants of the PfMSP142 protein from infected A549 cells were preferentially associated with the cells and not secreted into the media when examined by immunoblotting. One variant of PfMSP142, PfMSP142 (DSA), which contained the decay-accelerating factor (DAF) signal sequence and GPI anchor domain was shown to preferentially associate with the cell surface by FACS analysis using the 5.2 mAb. These vectors are currently being evaluated for their capacity to induce antibody and T cell responses to the PfAMA1 and PfMSP142 antigens. Results of this analysis will be presented.

CARDIOVASCULAR GENE THERAPY 21. Peptide-Targeted Ad19p-Based Adenoviral Vectors for Renal Gene Delivery Laura Denby,1 Lorraine M. Work,1 Dan J. Von Seggern,2 Eugene Wu,2 Ela Hunt,3 Stuart A. Nicklin,1 Andrew H. Baker.1 1 BHF Glasgow Cardiovascular Research Centre, University of Glasgow, Glasgow, United Kingdom; 2Department of Immunology, Scripps Research Institute, San Diego, CA; 3 Bioinformatics Research Centre, University of Glasgow, Glasgow, United Kingdom. Systemic administration of gene delivery vectors resulting in renalselective transduction would have significant implications for therapy of kidney diseases. To identify kidney-targeting peptides we used in vivo phage display with intravenous administration of a M13 phage library into 12 week old, male Wistar Kyoto (WKY) rats. Following 3 rounds of in vivo biopanning 307 phage inserts were sequenced and 3 consensus peptides identified – APASLYN (APA), HITSLLS (HIT) and HTTHREP (HTT). Homogeneous phage populations were amplified and infused into rats and the level of phage accumulation in the kidney determined. Each peptide demonstrated increased accumulation in the kidney compared to a control, insertless phage (APA 255-fold increase; HIT 152-fold increase; HTT 43-fold increase vs control insertless phage). Our previous studies identified an adenovirus serotype 5 capsid pseudotyped with serotype 19p fibers (Ad19p) as a candidate platform vector for re-targeting due to its lack of native hepatic Molecular Therapy Volume 13, Supplement 1, May 2006 Copyright  The American Society of Gene Therapy

tropism (Denby et al, 2004). Each candidate kidney-targeting peptide (APA, HIT, HTT) was genetically incorporated into the HI loop of the fiber of Ad19p between amino acids 331 and 332. Initially, transduction with the peptide modified vectors was assessed in vitro in rat cell lines. Peptide-modified Ad19p (APA, HIT and HTT) produced a significant increase (8- to 24-fold increase versus unmodified Ad19p; p<0.05) in transduction of rat glomerular endothelial cells in a dose-dependent manner. Transduction was restricted to the renal cell line with no elevation in gene delivery evident in either rat hepatocytes (Arl-6) or rat prostate endothelial cells (Y-PEN) compared to non-modified Ad19p. In vivo transduction was assessed in 8 week old, male WKY rats administered 3.5 x 1011 viral particles of peptide-modified or control Ad19p and sacrificed 5 days post-virus delivery. Immunohistochemical staining of tissues demonstrated gene expression in the kidney of those animals infused with the peptide-modified Ad19p but not the unmodified control. Furthermore, each vector had a different distribution pattern with the HTT vector transducing collecting duct cells and the APA and HIT vectors predominantly transducing glomerular endothelial cells. Combining phage display targeting peptides with the novel Ad19p platform vector resulted in renal-targeting viral vectors via systemic administration. These studies have important implications for genetherapy based intervention in the treatment of renal disease. Denby et al (2004) - Adenoviral serotype 5 vectors pseudotyped with fibers from subgroup D show modified tropism in vitro and in vivo. Human Gene Therapy, 15: 1054-1064.

22. Creation of a Biological Pacemaker by Cell Fusion Hee Cheol Cho, Yuji Kashiwakura, Eduardo Marbán. 1 Department of Medicine, Institute of Cardiobiology, Baltimore, MD. As an alternative strategy to electronic pacemakers or to gene therapy/stem cell approaches, we explored the feasibility of converting ventricular myocytes into pacemakers by cell fusion. The idea is to create chemically-induced heterokaryons between ventricular myocytes and syngeneic fibroblasts engineered to express pacemaker ion channels, HCN1. In order to examine fusion events, guinea pig (gp) fibroblasts stably-expressing HCN1 channels encoding the pacemaker current, If, were fused with freshly-isolated guinea-pig ventricular myocytes using polyethylene glycol (PEG 1500). Within 3 minutes of dehydration and rehydration, the gp fibroblasts with GFP as a reporter fused with ventricular myocytes as verified by the sudden introduction of GFP fluorescence into the myocytes. Current-clamp of fused myocyte-fibroblast cells exhibited spontaneous action potentials with a slow phase-4 depolarization. Subsequent voltageclamp recordings with 2 mM external Ba2+ to block IK1 revealed the heterologously expressed If, which was not detectable either in ventricular myocytes alone or in myocytes fused with control fibroblasts expressing only GFP. Equipped with these data, we focally-injected the HCN1-expressing gp fibroblasts suspended in 40% PEG 1500 into the apex of gp heart. Electrocardiograms taken 3-6 days after the injection revealed ectopic ventricular beats that were identical in polarity and similar in morphology to those recorded during bipolar pace-mapping of the apex in the same animal (n = 5 of 13). These ectopic beats were not observed in animals injected with control fibroblasts (n = 7). Furthermore, single ventricular myocytes were isolated from the site of cell-injection to measure the amount of pacemaker currents from the heterokaryons. Freshly isolated heterokaryons formed by fusion between myocytes and fibroblasts expressing HCN1-GFP expressed pacemaker current density of -12 ± 2 pA/pF at -130 mV (n=9), which is comparable to the reported values of pacemaker density in sinoatrial nodal cells. Control heterokaryons formed by fusion between myocytes and fibroblasts S9