410 Adenovirus Mediated NIS Expression in Xenografted Prostate Tumors Requires a Minimal Threshold of Viral Dose for Efficient Radioiodine Uptake Molecular Therapy Volume 19, Supplement 1, May 2011 Co[.]
ADENOVIRUS AND OTHER DNA VIRUS VECTORS II the function of AAPs derived from various serotypes The current system reported in literature relies on mouse monoclonal antibody (mAb) A20, which reacts specifically with assembled AAV2 particles by recognizing an epitope(s) that is present only in the assembled but not in the unassembled VP proteins Although the system based on mAbs against intact AAV particles is reliable and straightforward, the availability of such mAbs for serotypes other than AAV2 is currently very limited; therefore, it would not be easy to expand its application to various serotypes To overcome this limitation, we constructed an AAV capsid assembly reporter, AAV-Rep2, and sought to exploit this reporter to indirectly assess the AAV capsid assembly AAV-Rep2 carried only the AAV2 rep gene between the two AAV2 inverted terminal repeats; therefore it could express Rep proteins but required the cap gene expression in trans for its viral genomes to be packaged in virion shells HEK293 cells were co-transfected with pAAV-Rep2 (a plasmid containing the AAV-Rep2 genome), pCMV-AAV9-VP3 (a plasmid expressing only AAV9-VP3) and an adenovirus helper plasmid in the presence or absence of the fourth plasmid expressing AAP2 (the reported AAP derived from AAV2) or AAP9 (a putative AAP derived from AAV9) At 48-hours post-transfection, DNase I-resistant AAV-Rep2 viral genomes were quantified by a dot blot assay As a result, our new system could identify the functional AAP9 amino-acid sequence and successfully demonstrate the requirement of AAP expression in efficient AAV9 capsid assembly and the ability to complement the function of an AAP derived from one serotype with that from another Thus, the new system we report here provides a powerful and reliable means to study the function of AAPs derived from various serotypes and serves as a tool for AAV capsid engineering that disrupts the AAP ORFs 408 Directed Evolution of Adeno-Associated Virus: Construction of an Enriched Random Insertion Library and Artificial Selection To Identify Structure-Function Relationships Justin H Judd,1 Jonathan Silberg,1,2 Junghae Suh.1 Bioengineering, Rice University, Houston, TX; 2Biochemistry and Cell Biology, Rice University, Houston, TX Adeno-associated virus (AAV) is a promising clinical therapeutic gene delivery vector due to its unusually high safety profile Still, virus targeting is a critical parameter in successful gene delivery that has yet to be entirely resolved Using the crystal structure of AAV2 as a structural guide, rational design has been used to achieve vector targeting through ligand-receptor interactions However, detailed a priori knowledge of protein structure and function is still elusive, often precluding direct translation of known cell-targeting ligands into the context of the AAV capsid Directed evolution is an attractive alternative for altering virus tropism for which success has been reported using mutagenic techniques such as error-prone PCR and gene shuffling Transposon-based random insertion or random domain insertion by DNAse can be used to identify ideal insertion sites for known targeting ligands However, limitations to these approaches include a high frequency of frameshifted, non-viable mutants and/ or limited crossover diversity, reducing the output of ensuing, often tedious, selection efforts To address these vector design challenges, we have developed a versatile, enriched, random insertion library using the AAV2 capsid gene (cap) as a scaffold Briefly, a plasmid carrying cap was randomly linearized, blunted and ligated to a linker containing directional, unique, flanking restriction sites encoding small amino acids Cap genes with a single insert were isolated and non-viable mutants were removed from the pool Remaining cap genes - containing a single, in-frame, correctly oriented, randomly inserted linker - were subcloned into an AAV expression vector and the resultant library named pAAV_RaPID (Random Peptide Insertion by DNase) The pAAV_RaPID plasmid library can be used to quickly insert any peptide motif randomly into the AAV cap gene S158 with a high frequency of viable variants and broad crossover which should increase the occurrence of functional AAV variants in directed evolution studies Additionally, we demonstrate that the carefully selected restrictions sites used in the construction of the pAAV_RaPID library facilitate a novel vector-religation/recombination event, resulting in a high-throughput scanning alanine substitution library with enhanced diversity Furthermore, we show the utility of this latter library variation in quickly identifying important functional motifs in the AAV2 capsid protein using simple in vitro selection methods Adenovirus and Other DNA Virus Vectors II 409 Necrosis and Autophagy, but Not Apoptosis, Are Induced in Macrophages In Vivo upon Interaction with Adenovirus Nelson C Di Paolo,1 Dmitry M Shayakhmetov.1 Medical Genetics, University of Washington, Seattle, WA Several homeostatic and pathological cell death mechanisms exist, including apoptosis, pyroptosis, necrosis, and others We are interested in understanding the cell death pathway induced in macrophages in vivo in response to a model virus, adenovirus (Ad) Our studies in mouse models revealed that splenic methallophilic CD169- and MARCO-positive marginal zone macrophages (MZMø) selectively trap Ad after intravascular inoculation After interaction with Ad, these cells undergo a rapid, caspase-independent pro-inflammatory cell death that is distinct from classical apoptosis Staining of spleen sections with TUNEL or activated caspase-3-specific antibody revealed that MZMø undergo a caspase-3-independent type of cell death However, injection of mice with propidium iodine (PI) revealed numerous cells with PI-positive nuclei in splenic MZ 1h after virus administration, indicating that the plasma membrane integrity was compromised, a hallmark of necrotic type of cell death Furthermore, we found that Ad entry into MZMø induces formation of LC3-positive autophagosomes Electron microscopy studies showed that MZMø containing virus particles have complete disorganization of the cytoplasm and swelling and breakdown of mitochondria and other organelles by 4h after Ad challenge Interestingly, administration of a mutant Ad, Ad-ts1, which is unable to rupture cellular endosomes, showed that both CD169- and MARCO-positive cells survived in the splenic marginal zone after virus injection, suggesting a link between endosome rupture and the induction of necrotic type cell death in macrophages These results shed light on the unique mechanism of the adenovirus-induced macrophage death, as well as broaden our understanding of the biology of adenovirus that is being intensely studied for vaccination and as cancer therapeutic 410 Adenovirus-Mediated NIS Expression in Xenografted Prostate Tumors Requires a Minimal Threshold of Viral Dose for Efficient Radioiodine Uptake Miguel A Trujillo,1 Michael J Oneal,2 Samantha McDonough,1 John C Morris.1 Endocrinology, Mayo Clinic Rochester, Rochester, MN; Molecular Medicine, Mayo Clinic Rochester, Rochester, MN Prostate cancer (PCa) remains the most common malignancy in men, with an estimated 217,730 new cases and 32,050 deaths in the United States in 2010 Despite new aggressive approaches, there is a high rate of recurrence Gene therapy/virotherapy (GTV) strategies represent a rational direction for the development of novel therapeutics However, the parameters required for effective GTV such as viral replication, spread, and gene transfer efficiency are not well validated Clinical trials have demonstrated extremely limited tumor transduction, and this limitation remains the fundamental Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy ADENOVIRUS AND OTHER DNA VIRUS VECTORS II barrier to effective cancer gene therapy The efficiency issue has been historically addressed by increasing the administration dose One conceptual approach to improve limited infectivity is to permit local vector replication after delivery We reason however that there should be a minimum viral dose threshold required for efficient tumor transduction To test this hypothesis we LnCaP tumor xenografts intratumorally with the conditionally replicating adenovirus Ad5PB_ RSV_NIS CRAd in which the E1a gene is driven by the prostate specific promoter for Probasin and the cassette RSV promoter-human NIScDNA-bGH polyA replaces the E3 region (CRAd Ad5PB_RSVNIS) In vivo NIS expression, as a marker for viral transduction, was measured by 99Tc uptake and imaging A kinetic curve of 99Tc uptake was constructed showing quantifiable isotope uptake for up to one month post-infection These data indicate that the Ad5PB_RSV-NIS CRAd induces long term expression of functional NIS in LnCaP tumors in vivo and, given the kinetics of NIS expression, suggest that Ad5PB_RSV-NIS replicates in vivo The viral-dose response curve shows that a minimum dose of 1010 vp/tumor is required for quantitative radioisotope uptake Taken together, our data indicate that, despite viral replication, a minimal injected viral dose threshold is required for efficient tumor transduction These results also predict that CRAds mediated GTV will also require a threshold intratumoral viral dose for efficacy 411 Cancer Specific Peptide-Tagged Ad Vectors in Systemic Therapy of Aggressive Solid Tumors Anke Schmidt,1 Christian Eipel,2 Katharina Fürst,1 Nadine Sommer,1 Jens Pahnke,3 Brigitte M Pützer.1 Department of Vectorology and Experimental Gene Therapy, Biomedical Research Center, University of Rostock, Rostock, Germany; 2Institute of Experimental Surgery, University of Rostock, Rostock, Germany; 3Neurodegeneration Research Lab, Clinic of Neurology, University of Rostock, Rostock, Germany Significant advantage of targeted anticancer treatment consists in the possibility to restrict maximum therapeutic efficacy to the malignant cell population by reducing toxicity in healthy tissues Using different clinical models for aggressive medullary thyroid carcinoma (MTC), we have recently identified peptide ligands that bind highly selective to tumor cells By linking the most convincing SRESPHP peptide to an adenoviral (Ad) vector expressing the MTCrelated oncogene inhibitor RET∆TK, gene transfer was specifically directed to neoplastic tissue after systemic virus administration We show that peptide-mediated delivery of RET∆TK significantly enhanced apoptosis, resulting in a strong inhibition of orthotopic and xenograft tumor growth Conversely, tumors treated with controls expanded their initial size without notable cell death According to the therapeutic effect, strong virus accumulation was found exclusively in thyroid carcinomas Strikingly, application of native tropism depleted viral vector linked to tumor-selective peptide was accompanied by a substantial reduction of Ad binding to the liver Of note, single systemic injection of a low dose (10e8 pfu/mouse) of MTC-specific Ad.RET∆TK induced regression of multiple tumors at different sites in all treated animals In sum, our results open up the possibility for an efficient cancer cell-specific therapy of primary MTC, their migrating populations and potentially metastases This work was supported by DFG grants PU 188/5-1/5-2, PU 188/5-3, and the FORUN program (grant 889017) of Rostock University Medical Faculty Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy 412 Enhancing Therapeutic Effects of Oncolytic Agents through Switching of Mechanism of Tumor Killing Hannah Chen,1 Padma Sampath,1 Wei Hou,1 Rachel Sikorski,1 Stephen H Thorne.1 Surgical Oncology, University of Pittsburgh, Pittsburgh One advantage of oncolytic viral therapies is their ability to destroy tumor cells through multiple mechanisms of action, including direct oncolysis, immune activation and vascular collapse However, a careful balance is often required as many approaches that enhance immunotherapeutic potential of the vectors result in reduced oncolysis due to premature viral clearance from the tumor We have therefore looked to create ‘modular’ vectors where the mechanism of action can be switched from predominantly oncolytic to predominantly immunotherapeutic through exogenous application of a small molecule external regulator Modulation of gene expression from oncolytic viruses or gene therapy vectors is often desirable for the purpose of controlling therapeutic gene delivery, control of immune response, or as a means of rendering viral replication conditional, as a safety mechanism We have focused on vaccinia virus, as a potent oncolytic agent currently demonstrating significant clinical potential A novel strategy to regulate protein stability was applied, involving use of a small protein domain termed a destabilizing domain (DD) that can be cloned into either viral genes or transgenes This confers instability to the protein of interest (POI) that may be reversed upon the exogenous administration of a small molecule that binds specifically to the DD Using this strategy, we have demonstrated conditional production/destruction of various transgene products expressed from vaccinia virus, including reporter proteins, viral proteins and therapeutic molecules such as immunomodulatory cytokines both in vitro and in vivo Enhanced anti-tumor activity can be achieved in mouse tumor models following systemic delivery of oncolytic vaccinia viruses harboring conditionally regulated cytotoxic or immunotherapeutic proteins, demonstrating the potential of this strategy to augment oncolytic virotherapy by offering reversible control of the timing and level of specific protein activity In addition, a stabilizing domain (SD) that works through opposing regulation of the POI via binding of the same small molecule has been developed, allowing opposing regulation of multiple gene products and ‘switching’ of viral mechanism of action This technology can be extended to other gene therapy applications and provides a novel and clinically relevant method for regulating the functional output of various protein targets 413 A Novel Adenoviral Hybrid-Vector System Carrying a Plasmid Replicon for Safe and Efficient Cell and Gene Therapeutic Applications Richard Voigtlander,1 Martin Hausl,1 Rudolf Haase,2 Armin Baiker,1 Anja Ehrhardt.1 Virology, Max von Pettenkofer-Institute / LMU, Munich, Germany; 2Pharmacy, LMU, Munich, Germany In dividing cells the two aims a gene therapy should accomplish are defined as the nuclear distribution and retention of therapeutic DNA Because monosystems fail to fulfil both tasks with equal efficiency, hybrid-vector systems are promising in facing the challenges of molecular medicine Our hybrid-vector system HDAdV-pEPito synergizes the helper-dependent adenoviral vector (HDAdV) with the plasmid pEPito containing the therapeutic gene and a special DNA sequence called SMAR (Scaffold/Matrix Attachment Region) for episomal retention and replication Our technique provides a tool for stable maintenance of transgenes reducing the risk of insertional mutagenesis HDAdV-pEPito and the SMAR deleted control (HDAdV-pEPito-∆SMAR) were generated with our laboratory’s novel BAC-technology Both vectors carry the respective linearized S159 ... promoter-human NIScDNA-bGH polyA replaces the E3 region (CRAd Ad5PB_RSVNIS) In vivo NIS expression, as a marker for viral transduction, was measured by 99Tc uptake and imaging A kinetic curve of 99Tc uptake. .. required for quantitative radioisotope uptake Taken together, our data indicate that, despite viral replication, a minimal injected viral dose threshold is required for efficient tumor transduction... System Carrying a Plasmid Replicon for Safe and Efficient Cell and Gene Therapeutic Applications Richard Voigtlander,1 Martin Hausl,1 Rudolf Haase,2 Armin Baiker,1 Anja Ehrhardt.1 Virology, Max von