ADENOVIRUS VECTORS: VECTOR TECHNOLOGIES downstream, of the transgene The selection of these fragments was based on a series of gel shift assays following incubation of nuclear protein extracts with different MoMLV sequences Production of both vectors was efficient and comparable to that obtained with first generation adenoviral vectors (∼1012 - 1013 particles/ml by QPCR) To evaluate if these constructs were able to integrate into genome, the rat submandibular gland A5 cell line was transduced with AdLTR2EF1a-EGFP at 100 particles/cell After 10 days, colonies were counted and 8.1% (of 869) exhibited green fluorescence while none of the colonies (∼ 900 each) obtained after similar transduction with two conventional adenoviral vectors, AdCMVEGFP and AdEF1a-EGFP, exhibited green fluorescence PCR assays on DNA from the cloned cells transduced with AdLTR2EF1a-EGFP were consistent with possible transgene integration into genomic DNA To test if these constructs could mediate long-term transgene expression in vivo, AdLTR2EF1a-hEPO was delivered into rat submandibular glands at 109 particles/gland (only one gland targeted) Three of rats showed elevated hEPO and hematocrit levels for two months, and in one rat elevated hEPO and hematocrit levels persisted until the end of the experiment (5 months) Transduction with the conventional adenoviral vectors, AdCMV-hEPO and AdEF1a-hEPO, resulted in elevated hEPO levels for ∼ week PCR assays indicated that after AdLTR 2EF1a-hEPO transduction, the EF1a-hEPO sequence was found in extracted DNA at months endpoint from targeted submandibular glands of all three rats expressing hEPO for at least two months In two of these samples, PCR assays also suggested that an integration event occurred We detected anti-hEPO antibody in sera from the two rats whose hEPO levels decreased after two months However, in one rat with persistent high levels of hEPO for months, there was no evidence of anti-hEPO antibody Together, these results suggest that this second generation hybrid adenoretroviral vector, which is more efficiently produced than the original hybrid vector, can mediate long-term transgene expression in vivo trimerization is not necessary for the heat-stability phenotype (1) To facilitate pseudotyping adenoviral vectors with pIX variants we have set up an efficient system for the generation of pIX-expressing helper cell lines With an optimized pIX-expression cassette, we generated monoclonal and polyclonal helper cell lines that express wt as well as modified pIX genes to levels equivalent as in a wt HAdV-5 infected cells Immune-affinity electron microscopy on viruses lacking the pIX gene demonstrated that more than 96% of the particles contain pIX protein incorporated in their capsids after propagation on the pIX-expressing helper cell lines In addition, the pIX amounts in the helper cells are sufficient to generate heat-stable particles Finally, the ratio between the pIX protein and other capsid proteins is identical to those found in wt particles The cell lines are very stable: cell lines grown for two months in the absence of selection contain pIX in amounts identical to the parental line This demonstrates that pIX is not toxic to cells These data, together with our previous demonstration that alpha-helical spacers can be fused to pIX to expose new ligands on the outer surface of particles(2), establishes that modification of pIX is feasible for pseudotyping adenovirus vectors Vellinga, J et al (2005) The coiled-coil domain of the adenovirus type protein IX is dispensable for capsid incorporation and thermostability J.Virol (in press) Vellinga, J et al (2004) Spacers increase the accessibility of peptide ligands linked to the carboxyl terminus of the adenovirus minor capsid protein IX J.Virol 78: 3470-9 878 Toward ‘Pseudotyping’ Adenovirus Vectors: Efficient Generation of pIX-Expressing Helper Cell Lines Background Cellular uptake of group C adenoviruses critically depends on the availability of the cognate Coxsackie Adenovirus Receptors (CARs) on the cell surface In cardiovascular tissues paucity of CARs is a limiting factor for the successful application of adenoviral vectors Additionally, immune responses elicited by Ad infection diminish transgene expression due to the elimination of free Ad and transduced cells Polymer modification of adenoviral surface potentially addresses these problems by masking Ad particles from neutralizing antibodies and providing a convenient platform for the conjugation of ligands possessing transduction-facilitating properties We report here that a covalent modification of Ad surface with a photoactivatable polymer derivatized with protein transduction domains (PTD), TAT and Antp, increases transduction efficiency Methods A positively charged, thiol-reactive, photoactivatable polyallylamine (PAA) derivative was coupled with TAT-Cys and Antp-Cys Ad samples suspended in water or PBS of different ionic strength (x1 and x5) were admixed with PTD-derivatized and unmodified polymers to obtain final polymer/Ad ratios ranging from 1:100 to 1:5 (w/w) Initial charge-based polymer/virus attachment was rendered covalent by 15 sec UV light (350 nm) illumination Surface charge and size of the Ad/polymer complexes were measured by zeta-potentiometry and dynamic light scattering, respectively Ad surface modification was examined by transmission electron microscopy (TEM) employing colloidal anionic gold Transduction of a rat aortic smooth muscle cell line (A10) and primary bovine aortic endothelial cells (BAEC) was assessed by the fluorescence microscopy and the fluorimetry of cell lysates In some experiments the cells were pretreated with the recombinant soluble knob protein (5 µg/ml) prior to the Ad infection Rob C Hoeben,1 Taco G Uil,1 Jeroen De Vrij,1 Leif Lindholm,2 Jort Vellinga.1 Dept of Molecular Cell Biology, Leiden University Medical Center, Leiden, Netherlands; 2Got-a-Gene AB, Stena Center 1B, Gothenburg, Sweden Specificity of gene delivery is a key prerequisite for gene therapy We have previously demonstrated that modification of the minor capsid protein IX (pIX) can be used to modify cell type specificity of human adenovirus type-5 (HAdV-5) particles The 14.3 kDa hexon-associated pIX is strongly conserved between the different members of the mastadenoviruses Protein IX is believed to function as a capsid cement by linking the hexons in groups-of-nine (GONs) Although viruses lacking pIX not form GONs, and are less heatstable than wild-type viruses, they can be propagated with the same kinetics and yields as the wild-type viruses Deletion mutants lacking (parts of) the conserved N-terminal domain are not incorporated into the virion, implicating this domain in capsid formation In contrast, pIX molecules with mutations in other conserved domains (i.e the central alanine stretch, and the putative leucine-zipper domain) are efficiently incorporated into virions The carboxyl terminal domain contains the putative leucine-zipper domain that may be responsible for trimerization via the formation of a coiled-coil structure Mutants in this region are efficiently incorporated into the capsid, suggesting that trimerization is not essential for capsid formation Further analysis of these mutants revealed that deletion of the putative leucine-zipper domain does not affect capsid stabilization by pIX, demonstrating that S340 879 Transduction Enhancement by the Adenovirus/Polymer Complexes Derivatized with Protein Transduction Domain Peptides Ilia Fishbein,1 Ivan S Alferiev,1 Richard Gaster,1 Michael Chorny,1 Origene Nyanguile,1 Robert J Levy.1 Cardiology, The Children’s Hospital of Philadelphia, Philadelphia, PA Molecular Therapy Volume 11, Supplement 1, May 2005 Copyright The American Society of Gene Therapy ADENOVIRUS VECTORS: VECTOR TECHNOLOGIES Results Association of Ad with unmodified polymer increased surface charge of the vector from -31.67 mv to -11.89 mv, while the interaction with the Antp- and TAT-modified polymers inverted the surface charge to +3.75 mv and +12.09 mv, respectively Recharging of the Ad surface was confirmed by the TEM demonstrating the attraction of nm anionic gold particles to the surface of the Ad modified with TAT-derivatized polymer, but not to the surface of naive Ad The association of Ad-GFP with PTDderivatized polymers resulted in 3-7-fold increase of GFPexpression in cells, whereas unmodified polymer-derivatized virus demonstrated the same efficiency as the naive Ad Pretreatment of A10 cells with knob protein caused 86% transduction inhibition with free and unmodified polymer-derivatized Ad, while the transgene expression of the PTD/polymer-modified Ad was not affected Optimization of the transduction protocol revealed maximal enhancement of transgene expression at a polymer/Ad ratio of 1:10 Also, complexes prepared in x5 PBS achieved higher GFP expression than those prepared in water or x1 PBS, which correlated with smaller size of complexes (330 nm vs 1360 and 1060 nm diameter, respectively) Conclusion Covalent attachment of PDT-decorated polymers to the Ad surface increases transduction in vitro 880 AAV-Rep 78 Mediated Rescue of a 27kb Transgene Cassette from a Helper-Dependent Ad Vector Genome Hongjie Wang,1 Dmitry Shayakhmetov,1 Andre Lieber.1 Medical Genetics, University of Washington, Seattle, WA AAV Rep78 is thought to increase site-specific integration of AAV vectors into the AAVS1 site within human chromosome 19 We have constructed helper-dependent (HD) adenovirus vectors containing a 27kb globin LCR-GFP expression cassette flanked by AAV ITRs (HD-Ad.AAV-GFP) Our goal was to test whether transient Rep78 expression would rescue this large expression cassette from the HD-Ad genome and increase its site-specific integration We planned to use HD-Ad5/35 vectors to deliver the rep78 gene to our target cells, MO7e cells, which are a human erythroleukemic cell line Construction of Rep78-expressing Ad vectors is problematic, however, because trace amounts of Rep78 inhibit Ad replication We therefore placed the rep78 gene under the control of the β-globin promoter, which was found (in preliminary studies) to have low activity in 293 (producer) cells but high activity in target MO7e cells We produced this vector (HD-Ad.LCR-rep78) at titers of 4.1x1012 genomes per ml Western blot analysis with Rep-specific antibodies confirmed rep78 expression from HDAd.LCR-rep78 We then performed rescue assays in MO7e cells by infection with HD-Ad.LCR-rep78 and HD-Ad.AAV-GFP While no rescue of the AAV-LCR-GFP cassette was detectable by Southern blot analysis upon infection with the two HD-vectors, addition of a first-generation Ad vector caused rescue This effect was nullified by addition of hydroxyurea, an inhibitor of adenoviral DNA replication We drew the conclusions that a large (∼27kb) transgene cassette can be excised from HD-Ad genomes by Rep78, and E1deleted first-generation vectors are able to replicate their DNA and the DNA of co-infected HD-Ad vectors in MO7e cells, and Rep78 mediated rescue in MO7e cells requires Ad proteins and/or viral replication of HD-Ad.AAV genomes We are currently attempting to dissect these two possibilities We also performed transduction studies with HD-Ad.LCR-rep78 and HD-Ad.AAV-GFP vector Coinfection with HD-Ad.LCR-rep78 increased the overall stable transduction frequency of HD.Ad.AAV-LCR in MO7e cells about two-fold We are currently analyzing the integration frequency into AAV-S1 in the presence of HD-Ad.LCR-rep78 and the results will be presented Molecular Therapy Volume 11, Supplement 1, May 2005 Copyright The American Society of Gene Therapy 881 Development of Novel E1-Complementary Cells for Adenoviral Production Free of Replication Competent Adenovirus Deborah Farson,1 Lucy Tao,1 Derek Ko,1 Thomas Harding,1 Dominic Brignetti,1 De-Chao Yu,1 Yuanhao Li.1 Research & Development, Cell Genesys, Inc., South San Francisco, CA Historically adenoviruses, especially E1-deficient adenoviral vectors, are produced from E1-complementary cells such as human embryonic kidney 293 cells or human embryonic retinoblast PerC6 cells Each of these cell lines contain a fragment of the adenovirus genome that includes both the E1A and E1B coding regions, and have been shown to generate low levels of replication competent adenovirus (RCA) or other types of recombinants due to homologous recombination with cellular DNA We have developed a novel E1A/ 1B complementing cell line that is suitable for the large-scale production of adenovirus that does not generate RCA by separately inserting the coding sequences for E1A and E1B into different regions of the host cell genome Initially, retroviral vectors derived from Moloney Murine Leukemia Virus (MMLV) were separately constructed encoding the coding sequences for adenovirus E1A or E1B Retroviral supernatants were then prepared from these vectors and used to co-infect A549 cells, a human lung cancer cell line E1complementary clones were selected by a functional assay using an E1A-deficient adenovirus and two cell clones, clone 51 and clone 139 were chosen based upon viral yield Western blot analysis indicated that clone 51 and 139 express E1A, E1B 19kD and E1B 55kD proteins PCR and Southern blot were then used to demonstrate that the E1A and E1B genes are inserted into separate regions of the A549 genome Production of an E1-deleted adenovirus vector on the clones indicated that viral yields are comparable to 293 or PerC6 cells In comparison, production of E1-modified oncolytic adenoviruses was found to be superior for clones 51 and 139 than viral yields derived from 293 or PerC6 cells The E1complementation was stable over 10 weeks of continuous culture To conduct industrial scale adenoviral production for clinical application, clones 51 and 139 were adapted to serum-free suspension culture and shown to produce adenovirus at levels comparable to 293 suspension cells Most importantly, the adenoviral products from these cells were demonstrated to be free of RCA In a blind passage study, an RCA-free Ad-GMCSF virus was serially amplified for 20 passages on the new E1-clones, 293 and PerC6 cells The adenovirus from the last passage was then amplified in naïve A549 cells and assayed for RCA or other recombinants that cause cytopathic effect (CPE) The result indicates that both 293 and PerC6 cells produce recombinants that form CPE in A549 cells, whereas the new E1-clones not generate detectable recombinants by plaque assay or a PCR based recombination assay These new E1-complimenting cell clones should be of considerable use in the large-scale production of RCA-free adenovirus vectors for clinical application I own stock in Cell Genesys 882 Development of a Transposon-Based Approach to Identification of Novel Transgene Insertion Sites within Replicating Viral Genomes Peter Kretschmer,1 Fang Jin,1 Cecile Chartier,1 Paul Harden,1 Terry Hermiston.1 Gene Therapy Research, Berlex Biosciences, Richmond, CA Oncolytic viruses represent a potentially new treatment for cancer It is clear, however, that the potency of these agents must be increased, and one approach is to incorporate therapeutic genes that synergise with the lytic functions of the virus For Ad5, the choice of insertion site(s) is generally limited to a few known non-essential S341