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852 A Novel Genomic DNA Reporter Gene Model for the Study of Friedreich''''s Ataxia in Neuronal Cells Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy[.]
OTHER DNA VIRUSES Furthermore, in vivo mice biodistribution studies exhibited a decrease in liver sequestration of the hexon modified vectors Utilizing xenograft tumor models, anti-tumor efficacy of the hexon-modified CRAds was investigated There are several potential advantages of eliminating the Ad liver sequestration observed after intravenous injection, such as reducing hepatotoxicity and conceivably allowing more of the injected vectors to be available in the bloodstream to reach their desired tissue and tumor targets Whether these advantages hold true will require full clinical translation and evaluation 850 Design of Bi-Specific DARPin-Derived Adapters for Targeting of Adenovirus Vectors Galina Mikheeva,1 Birgit Dreier,2 Christian Hess,2 Edgar Boczek,2 Natalya Belousova,1 Andreas Plückthun,2 Victor Krasnykh.1 Department of Experimental Diagnostic Imaging, University of Texas M.D Anderson Cancer Center, Houston, TX; 2Department of Biochemistry, University of Zurich, Zurich, Switzerland Efficient and target-specific gene delivery in vivo is the top priority in development of genetic interventions for humans Because of the lack of natural specificity of viral gene vectors for tumors, the native tropism of these vectors should be modified This tropism modification often requires ablation of the virus’ specificity for its native receptor(s) and engineering of a novel tropism In this study we sought to develop bi-specific adapter molecules derived from the so-called designed ankyrin repeat proteins (DARPin) to direct human adenovirus serotype (Ad5) to Her2 receptor Such adapters would contain two types of DARPins: the Ad5-specific DARPin component that binds to Ad particles, and the anti-Her2 DARPin ligand that crosslinks the virus to the surface of target cells First, to select for anti-Ad5 DARPins that would bind to Ad virions with ablated natural tropism, a recombinant Ad5 fiber knob protein with impaired binding to CAR receptor was produced Next, this protein was used for the ribosome display-based selection of a highly diverse library of DARPin genes followed by affinity maturation This yielded the DARPin leads that bind the mutated Ad5 fiber knob with affinity in low nanomolar range Subsequent analysis of the complexes formed by these leads and the fiber knob established the stochiometry of this interaction These data guided the design of multi-valent adapters with improved stability of the virus-adapter complexes The adapters were made Her2-specific by fusing them with the previously developed anti-Her2 DARPin ligand and used to direct the CAR-ablated, reporter-expressing Ad5 vector to Her2-expressing cancer cells 851 Influence of Factor X on In Vitro and In Vivo Gene Delivery by Ad5 and Ad35 Vectors Jenny A Greig,1 Suzanne M K Buckley,2 Simon N Waddington,2 Alan L Parker,1 David Bhella,3 Rebecca Pink,3 Takashi Morita,4 Jerome Custers,5 Jaap Goudsmit,5 Stuart A Nicklin,1 John H McVey,6 Andrew H Baker.1 BHF Glasgow Cardiovascular Research Centre, University of Glasgow, Glasgow, United Kingdom; 2Department of Haematology, Royal Free and University College Medical School, London, United Kingdom; 3MRC Virology Unit, University of Glasgow, Glasgow, United Kingdom; 4Department of Biochemistry, Meiji Pharmaceutical University, Tokyo, Japan; 5Crucell, Leiden, Netherlands; 6Haemostasis and Thrombosis, MRC Clinical Sciences Centre, Imperial College London, London, United Kingdom Recently, the interaction between adenovirus (Ad) and the blood coagulation factor X (FX) was shown to be pivotal for liver transduction FX binds directly to the hexon of Ad5 and this interaction leads to hepatocyte transduction in vivo, an effect mediated through heparan sulphate proteoglycans Vectors based on the subspecies B Ads, including Ad35, are in development for gene therapy Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy as the Ad35 fiber uses the membrane protein CD46 as a high affinity cellular receptor instead of the coxsackie and adenovirus receptor, the primary receptor for the Ad5 fiber Based on this we investigated the Ad35:FX interaction in detail using Ad5, Ad35 and Ad35 chimeras: the pseudotype Ad5/F35, which contains the hexon and penton of Ad5 with the Ad35 fiber, and Ad5/P35/F35, which has the Ad5 hexon and the penton and fiber of Ad35 By electron cryomicroscopy at 31Å resolution we observed that FX bound to the Ad35 hexon The ability of the chimeric vectors and Ad35 to bind FX was analysed by BIAcore surface plasmon resonance (SPR) and compared to previous data characterising the Ad5:FX interaction SPR analysis revealed that Ad35 and the chimeric vectors bound to FX with high affinity: in particular Ad35 had an equilibrium dissociation constant (KD) of 5.18x10-8M in comparison to Ad5, KD of 1.83x10-9M In CHO-CD46 cells the presence of FX significantly inhibited transduction of vectors containing Ad35 fibers by 352-, 101- and 188-fold for Ad5/F35, Ad5/ P35/F35 and Ad35, respectively, an effect rescued by the addition of X-bp To assess targeting in vivo the vectors were injected into CD46 transgenic mice in the presence and absence of FX binding protein (X-bp) Localisation of the luciferase expressing vectors was visualised by whole-body bioluminescence and vector genomes were quantified by qRT-PCR In the absence of X-bp, Ad5/F35 and Ad5/P35/F35 exhibited high levels of lung vector accumulation with 5.6x105 and 5.7x105 vector genomes (VG)/50ng of total DNA isolated, respectively, whereas Ad5 demonstrated very poor lung targeting at 4.6x104 VG/50ng DNA Pre-administration of X-bp significantly reduced genome accumulation in lung by Ad5/F35 and Ad5/P35/ F35 1.9- and 6.6-fold, respectively However, lung transduction by Ad35 was significantly enhanced by 1.6-fold in the presence of X-bp Liver transduction by all vectors was significantly reduced by X-bp, indicating that FX has an effect on both the Ad5 and Ad35 hexons Our study shows that FX limits transduction through CD46 in vitro, which can be rescued by the addition of X-bp However, in vivo ablation of the FX interaction by X-bp enhances CD46-dependent transduction and genome accumulation by Ad35 Other DNA Viruses 852 A Novel Genomic DNA-Reporter Gene Model for the Study of Friedreich’s Ataxia in Neuronal Cells Michele M P Lufino,1 Javier Alegre-Abarrategui,1 Filip Lim,2 Richard Wade-Martins.1 Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, Oxfordshire, United Kingdom; 2Departamento de Biología Molecular, Centro de Biología Molecular “Severo Ochoa” (CSIC-UAM), Universidad Autónoma de Madrid, Madrid, Spain Friedreich’s Ataxia (FA), the most common form of inherited ataxias, is characterized by a slow progressive neurodegeneration affecting mainly the central nervous system and the cardiac tissue FA is caused by an abnormal expansion of GAA repeats in intron 1, which results in a reduction of levels of the mitochondrial protein frataxin (FXN) There is currently no effective treatment able to alleviate the neurological degeneration The lack of suitable neuronal models for the study of both the wild-type and the mutated forms of frataxin has hampered the understanding of its function and the development of therapeutic applications Here we describe the use of the infectious bacterial artificial chromosome (iBAC) technology, based on the herpes simplex virus type (HSV-1) amplicon vector, as part of a new improved model for the analysis of frataxin expression in neuronal cells We constructed iBAC-FRDA-Luciferase, a genomic DNAfusion reporter vector by modifying a BAC containing the entire 135 kb FRDA genomic DNA locus Using homologous recombination, we inserted seamlessly the luciferase sequence at the 3’ of the last exon of S325 OTHER DNA VIRUSES the FRDA gene (exon 5a) Using further homologous recombination we then subsequently introduced into intron ∼310 GAA repeats, amplified from FA patient’s DNA iBAC vector delivery into the neuronal cell line SH-SY5Y was achieved using HSV-1 amplicon system, which allowed high transduction of this cell line otherwise resistant to transfection procedures We demonstrated expression and detection of the fusion protein FXN-luciferase in SH-SY5Y and, most importantly, we show that the presence of GAA repeats in iBAC-FRDA-Luciferase induces repression of frataxin expression by 75%, recapitulating the inhibition of transcription observed in FA patient cells Further validation to this model came from preliminary experiments which showed that incubation with histone deacetylase (HDAC) inhibitors can relieve the repression generated by the expanded GAA repeats, which are believed to induce heterochromatin formation Current techniques for frataxin detection are laborious as they are based on RT-PCR and western blot techniques The new iBAC-FRDA-Luciferase genomic DNA expression vector described here represents a novel tool for the study of FA since it allows rapid quantitative analysis of FXN expression in different conditions in vitro and in vivo This vector will facilitate the study of frataxin function and will help unravel the complex mechanism by which expanded GAA triplets induce suppression of FXN expression The use of iBAC-FRDA-Luciferase could also provide a platform for high-throughput screening of drugs able to restore frataxin expression Finally injection in mouse brain can give insight on FXN delivery and expression in live animals, thus providing important information for future gene therapy applications Overall, the work represents a powerful and unique application for the very high transgene capacity (∼150 kb) of the HSV-1 amplicon system 853 Recombinant Parvovirus B19 Vectors: AAV2 Fivefold Channel Swap onto B19 Capsids Confers Increased Stability and Enhanced Transduction Efficiency to Chimeric B19 Vectors Kirsten A K Weigel-Van Aken,1 Yiwen Xiang,1 Linyuan Chen,1 Lakshmanan Govindasamy,2 Mavis Agbandje-McKenna.2 Pediatrics, Molecular Genetics & Microbiology, Powell Gene Therapy Center, Genetics Institute, Shands Cancer Center, University of Florida, Gainesville, FL; 2Biochemistry & Molecular Biology, University of FLorida, Gainesville, FL Parvoviral vectors have a great potential as gene therapy tools if they keep their promise of efficiency and safety Adeno-associated virus (AAV) serotype based vectors are currently being used in phase I and II clinical trials and some of the recently identified AAV serotypes bear promise for future use for specific indications One of the major challenges in parvoviral gene therapy remains vector targeting Parvovirus B19 is a human parvovirus with highly restricted tropism It replicates exclusively in human hematopoietic progenitor cells of the erythroid lineage and uses the blood group P antigen as primary receptor In an effort to exploit the selectivity and the bone marrow tropism of human parvovirus B19, we have generated recombinant B19 (rB19) vectors that encapsidate single-stranded AAV2 genomes into VP2 B19 capsids and have demonstrated that activated beta1 integrin functions as co-receptor for parvovirus B19 entry into human cells Due to its restricted tropism and the fact that the site for P antigen binding on the B19 capsid is known, allowing targeting peptide insertions, rB19 vectors have the potential for targeting non-erythroid cell types in the human bone marrow However, efforts to further develop these rB19-based vectors for gene therapy applications have been hampered by low packaging efficiency which must be overcome Based on previous reports that the fivefold channel in the capsid of AAV2 may play a role in Rep mediated genome encapsidation, we have generated chimeric capsids in which we replaced B19 fivefold channel forming residues with those of AAV2 (a B19-ch5AAV2 vector) with the goal of improving packaging S326 efficiency The fivefold channel swap had no effect on rB19 capsid assembly Wild-type B19 capsids are able to assemble and incorporate partial and full-length self-complementary (sc) AAV2 genomes In contrast, the B19-ch5AAV2 virions incorporated exclusively fulllength recombinant genomes suggesting that the presence of the AAV2 fivefold channel increased the DNA encapsidation efficiency Co-immunoprecipitation experiments revealed that B19-ch5AAV2 capsids interact with AAV2 Rep proteins, whereas no interaction was observed in B19 wild-type capsids In addition B19-ch5AAV2 vectors with packaged sc-Luc genomes were up to 12-fold more efficient in transducing HEK293 cells We also generated P antigen-binding site-deleted B19 capsids that express an EGFR-targeting epitope and demonstrated efficient targeting of EGFR+ breast cancer cells in vitro In summary, by swapping the AAV2 fivefold channel onto B19 capsids we have been successful in increasing the full-length genome encapsidation of pseudo-typed B19 vectors and have gained a substantial increase in transduction efficiency These novel rB19 vectors will be useful in exploring viral vector targeting to bone marrow-resident cells, such as EGFR+ breast cancer metastases 854 Methods To Enhance the Spread and Onclytic Activity of HSV Vectors in GBM Therapy Chang-Sook Hong,1 Ajay Niranjan,1 Bonnie Reinhart,2 Ian Pollack,1 Joseph C Glorioso,2 Paola Grandi.1 Department of Neurological Surgery, University of Pittsburgh School of Medicine, Pittsburgh, PA; 2Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA Oncolytic HSV (oHSV) vectors have shown promise in the treatment of patients with recurrent glioblastoma However, the great majority of patients show incomplete responses indicating that this treatment modality requires further development Impediments to effective therapy include limited vector distribution on delivery and inefficient vector replication within tumor mass Experiments were carried out to (i) examine novel mutant vector backbones with enhanced oncolytic activity relative to vectors commonly used in clinical trials and (ii) modify the extracullar matrix (ECM) in order to optimize vector distribution in the tumor mass without encouraging tumor cell migration A new class of oncolytic vector (JD0) deleted for the immediate early gene ICP0 in combination with removal of the joint sequences was shown to grow specifically in a variety of tumor cells JD0 also possessed a remarkable oncolytic activity following treatment of animal models of human glioma without toxicity for normal brain To enhance viral spread, we investigated the use of the matrix metalloproteinase (MMP9) as means to degrade collagen type IV, a common component of the ECM and basement membranes of the glioblastoma tumor but absent in normal brain tissue Tumor cell lines transduced for expression of MMP9 increased the distribution and infectivity of tumor spheroids in vitro with a consequent improvement in oncolytic activity The over expression of this protease also decreased the efficiency of migration of glioma cells in vitro Experiments are underway to determine whether the improved distribution pattern in vitro can be recapitulated in vivo using an MMP9 expressing human tumor model Success in these experiments will encourage the further development of oHSV that express genes encoding additional products to modify the tumor micro-environment in a manner to favor vector spread and interfere with tumor survival pathways Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy ... following treatment of animal models of human glioma without toxicity for normal brain To enhance viral spread, we investigated the use of the matrix metalloproteinase (MMP9) as means to degrade... of the AAV2 fivefold channel increased the DNA encapsidation efficiency Co-immunoprecipitation experiments revealed that B19-ch5AAV2 capsids interact with AAV2 Rep proteins, whereas no interaction... reports that the fivefold channel in the capsid of AAV2 may play a role in Rep mediated genome encapsidation, we have generated chimeric capsids in which we replaced B19 fivefold channel forming residues