764. Effective and Stable Gene Transfer into Human NK Cells Using an HIV-1-Based Lentiviral Vector System

RNA Virus Vectors II to cell fusion ultimately leading to cell death. We utilize the model to explore the impact of tumor architecture and the dynamics of tumor cell-virus interactions on the outcome of therapy. Methods: We simulated two patterns of tumor growth: a standard spatial growth model leading to compact three-dimensional tumor shapes, and a fractal-like tumor. In its compact form, whenever a tumor cell reproduced, it sampled the surrounding lattice space to fill a compartment if this was empty or ‘push’ a cell away. In the case of fractal tumor growth, each cell division is followed by a biased random walk and the cell occupies a space close to the parent cell. In both models, tumor growth is stochastic although the dynamics of tumor growth and resulting tumor architecture are different. The virus is introduced at localized regions of the tumor, where virus infection occurs with a given probability. Infected cells can fuse with surrounding cells with a variable probability and infected cells die with a probability that is higher than that of the untreated tumor. The simulation stopped when the population of uninfected cells reached a pre-defined carrying capacity. We performed 106 simulations for each set of parameters and summarized the results. Results: The starting tumor size at which infection takes place ranged from 10 to 90% of the tumor carrying capacity; subsequently, 1% of the tumor cell population was infected through virotherapy. The three major variables that influenced outcome were: initial tumor size, the rate with which infected cells died and the tumor architecture. Smaller tumors are more likely to be eliminated by the virus, irrespective of the architecture. Tumors with fractal architecture are significantly more difficult to eradicate and in such cases, viruses that slowly kill tumor cells are more effective. For a compact tumor, the sensitivity to the location of initial infection is less important compared to fractal-like tumors, for which a central infection is associated with a higher probability of eradication. Conclusions: The outcome of tumor virotherapy is highly variable. Success or failure depends on the size of the tumor at time of therapy, tumor architecture, and the rate with which infected cells die. Smaller tumors are more likely to be eradicated. Viruses that kill cells slowly are associated with a higher probability of successful therapy, a feature which proves crucial for success in fractal-like tumors. The optimal sites of infection of the tumor also depend on the architecture with opposite results for compact versus fractal tumors.

763. Toward Gene Therapy of Cancer: A Model for Selective Expression of Anti-Life and Pro-Death Transgenes

Agnes Holczbauer,1 Adriana Zingone,2 Kritika Kachapati,3 Suresh K. Arya.3,4 1 Laboraotry of Experimental Carcinigenesis, National Cancer Institute, National Institutes of Health, Bethesda, MD; 2Genetics Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD; 3Basic Research Laboratory, National Cnacer Institute, National Institutes of Health, Bethesda, MD; 4 Developmental Therapeutics Program, National Cnacer Institute, National Institutes of Health, Bethesda, MD.

To enhance effectiveness of gene therapy of cancer, we think it makes sense to target both cell survival and apoptosis pathways. As prototypes, we chose Bcl2 shRNA to down regulate cell survival and Bax transgene to up regulate apoptosis. To achieve cancer cell selectivity and yet maintain high level expression, we designed a transregulation model combining ideas from molecular virology and cancer biology. The model consists of driving the expression of Bcl2 shRNA and Bax transgene with HIV viral LTR, activate viral LTR with viral transactivator Tat, but drive viral Tat expression with cancer cell selective cellular hTERT promoter. The model requires that the LTR-gene X not be expressed without transactivation by Tat in target cells, that the hTERT promoter be able to drive Tat expression in these cells, and that Tat be able to activate LTR-gene X expression S286

in trans. We are now screening NCI 60 cell panel to test this model, with GFP as the indicator gene or gene X. The panel consists of cell lines derived from several of human cancer types, thus providing a spectrum of phenotypes. Of the cell lines tested so far, all but one met the above criteria – they are negative for GFP when transduced with the lentiviral GFP vector without an internal promoter (i.e. driven by the lentiviral LTR itself) and they display readily detectable GFP when co-transduced with the lentiviral Tat vector. One exception was that it did not support the expression of GFP from the GFP vector either with or without co-transduction with the Tat vector. This cell line could turn out to be valuable in shedding additional light on the mechanism and specificity of Tat tranactivation. Interestingly, we yet have not encountered a cell line form the panel where lentiviral GFP vector is constitutively expressed, that is cell line contains factor(s) that can complement viral Tat. While the picture might change when more cell lines and primary tumor cells are screened, so far the model holds true.

764. Effective and Stable Gene Transfer into Human NK Cells Using an HIV-1-Based Lentiviral Vector System

Su Su, Ramanathan Muthalagu, Duc M. Nguyen, Aleah Smith, Keyvan Keyvanfar, Sheila Rao, Andreas Lundqvist, Maria Berg, Richard Childs. 1 Hematology Branch, NHLBI/NIH, Bethesda, MD. Natural killer cells (NK cells) play an important role in innate immunity against tumors and viral infection. The development of an efficient method to genetically modify NK cells could be useful to genetically manipulate NK cells for therapeutic purposes and for studies characterizing NK cell tumor trafficking in vivo. Although HIV-1 based lentiviral vectors (LVs) have been used to efficiently transfer genes into human T-cells, little data exist on the use of LV vectors to transduce NK cells. In this study, we designed a variety of HIV-based LV vectors expressing enhanced green fluorescence protein (EGFP) controlled by different promoters (MSCV-LTR, sEF1a or human ubiquitin C) to transduce CD3- and CD56+ and/or CD16+ primary human NK cells. EGFP expression was measured by flow cytometry 3-4 days following LV transduction to assess transduction efficiencies (TE). Flow cytometric analysis of NK cells stained with propidium iodide revealed polybrene, an agent used to enhance TE, was highly toxic to NK cells, inducing up to 25% NK cell death after 8 hours of treatment at 8µg/mL. In contrast, exposure of NK cells to protamine sulfate at 8µg/ml for up to 16 hours enhanced LV TE without inducing obvious NK cell death. LVs with the MSCV-LTR promoter were found to mediate the most effective gene transfer to primary NK cells compared LVs with the sEF1a promoter or human ubiquitin C promoter. Using an EGFP LV driven by a MSCV-LTR promoter in the presence of protamine sulfate, we successfully transduced the natural killer cell line NKL, with long-term EGFP expression detected in up to 98% NKL cells. Using the same conditions, the following observations were made: 1) Freshly isolated NK cells were difficult to transduce, with TE in the range of only 2-17% 2) Culturing freshly isolated NK cells for 24 hours in the presence of IL-2 (500 U/mL) significantly enhanced LV TE, with 20-30% of NK cells obtained from 3 different donors expressing EGFP 3) For freshly isolated NK cells cultured in IL-2 with irradiated EBV-LCL feeder cells x 9 days, up to 55% TEs were obtained after a single or double round of transductions respectively 4) Retronectin significantly augmented LV transduction of human NK cells; NK cells cultured in retronectin-coated plates were transduced efficiently with a low MOI 5) and maintained EGFP expression and >90% viability 10 days following LV transduction 6) In contrast to un-transduced NK cells, the phenotype of LV-transduced NK cells did not change significantly . Conclusion: Retronectin significantly augments LV transduction of human NK cells with a low MOI. Using Molecular Therapy Volume 16, Supplement 1, May 2008 Copyright © The American Society of Gene Therapy

Development of AAV Vectors retronectin-coated plates and a 2 round LV transduction protocol, TE of > 55% can be achieved in ex vivo expanded NK cells. This study provides an efficient method to introduce transgenes with long-term expression into human NK cells without a deleterious effect on NK cell viability.

765. Detection of Replication Competent Lentivirus in HIV-Based Lentiviral Vectors

Patricia Huang, Rafat S. Khan, Landon G. Piluso, David Hsu, Larry A. Couture. 1 Center for Biomedicine & Genetics, Beckman Research Institute of City of Hope, Duarte, CA. Despite the progress made in engineering a self-inactivating lentiviral vector, which uses multiple plasmids and renders the vector replication-defective during the process of cell transduction, the potential for generating a replication competent vector remains a concern. As a cGMP production facility, we have produced more than 30 batches of lentiviral-based vector. Accordingly, we are developing an in-house, two tiered approach to RCL testing which uses a PCRbased pre-screening step (1x107 or 0.1ppm sensitivity) prior to the conventional amplification-indication assay. Since an important step in the generation of RCL is its ability to obtain a functional polymerase gene, genomic DNA from transduced cells is recovered and the detection of the conserved polymerase sequence in the proviral DNA is assayed using PCR. Subsequently, the conventional p24-based assay for the amplification of potential RCL and infection of naïve indicator cells is performed for final release. This approach allows a practical in-process assessment of the material to be made within the manufacturing setting before more value is added to the material with further costly testing.

766. A Carriage for Sleeping Beauty: Development of a Hybrid Lentivirus-Transposon Vector

Conrad Vink,1 Adrian Thrasher,1 Bobby Gaspar,1 Waseem Qasim. 1 Molecular Immunology Unit, UCL Institute of Child Health, London, United Kingdom. The Sleeping Beauty transposon is a mobile DNA element reconstructed from salmonid fish that is currently being investigated as a potential vector for gene therapy. Excision and reintegration of DNA flanked by transposon inverted repeats is catalysed by a transposase enzyme. As an integrating vector, Sleeping Beauty offers the potential for stable delivery of therapeutic transgenes to dividing cells. However, Sleeping Beauty transposons are unable to enter target cells unaided. To address this, we are developing a hybrid vector which combines the efficient cell and nuclear entry properties of nonintegrating HIV-1 vectors with the integration mechanism of Sleeping Beauty. This strategy will exclude any virus-derived sequences from integration into the host genome. The Sleeping Beauty transposon also integrates within genes less frequently than HIV-1 and shows little preference for regions of active transcription, which may reduce the risk of insertional mutagenesis and cell transformation. We pursued a cis strategy in which one non-integrating lentiviral vector encodes a neomycin resistance cassette flanked by transposon inverted repeats and a second vector provides expression of the Sleeping Beauty transposase SB11. The efficiency of transposition following plasmid transfection and non-integrating lentiviral infection of HeLa cells was compared. Nonintegrating lentiviral vectors expressing SB11 mediated highly effective transposition from transfected plasmids. Transposition was confirmed by recovery of transposon-chromosome junctions using ligation-mediated PCR. However, transposition from non-integrated lentiviral cDNA could not be detected despite transduction of cells with transposon lentivector at high multiplicities of infection (MOI). Quantitative PCR analysis following titration of Molecular Therapy Volume 16, Supplement 1, May 2008 Copyright © The American Society of Gene Therapy

both plasmid and lentivector template suggests that initial transposon copy number is a limiting factor to transposition following lentivector transduction. Transposition is difficult to detect below 400 transfected plasmids per cell, but high MOI lentivector transduction delivers around 20 copies per cell. To further investigate the behavior of Sleeping Beauty at low copy number, we have defined a high resolution three-dimensional ‘transposition surface’ by varying both transposon and transposase plasmid copy number over a wide range and measuring the resulting integration frequency. Our data indicate that transposition increases with transposon copy number over all values tested. By contrast, the transposition surface is highly sensitive to both over- and underexpression of transposase at low copy numbers, so accurate optimization of transposase expression is critical for maximizing efficiency. Understanding the transposition surface at low copy number has generic implications for both our current delivery strategy and other Sleeping Beauty delivery systems.

Development of AAV Vectors 767. A Rapid, General Platform To Identify Functional Capsid Regions of Any AdenoAssociated Virus Serotype

James T. Koerber,1 Yue Yang,1 David V. Schaffer.1 1 Chemical Engineering, University of California at Berkeley, Berkeley, CA. Adeno-associated viral (AAV) vectors have demonstrated considerable potential as gene delivery vectors to treat a broad range of diseases, including hemophilia and Parkinson’s. However, AAV has several shortcomings that currently limit its clinical translation, including pre-existing immunity, poor transduction of some cells, and infection of off-target cells. A greater understanding of viral structure/ function relationships would benefit both rational design and directed evolution approaches to engineer enhanced vectors. To date, more than 100 different natural variants of AAV have been isolated from both human and non-human tissues. The sequence variation in the viral capsid of these variants confers a broad range of gene delivery properties including binding to a variety of cell surface receptors such as heparan sulfate proteoglycan (HSPG), sialic acid, fibroblast growth factor receptor (FGFR), and platelet derived growth factor receptor (PDGFR). Furthermore, exhaustive rational mutagenesis techniques have successfully mapped regions of the AAV2 capsid which are involved in HSPG binding and putatively, FGFR binding. However, the lack of extensive structure/function knowledge of other AAV variants has hindered our understanding of the basic biology of AAV infection and ability to engineer more effective variants. Accordingly, we have developed a novel, rapid, high-throughput platform to identify regions of the capsid of any AAV serotype that are involved in a specific viral function. Specifically, we have generated highly diverse AAV libraries based on AAV5 and AAV6 through random mutagenesis of the cap gene and selected for mutants that are defective in specific steps of viral infection. For example, to map key residues involved in receptor binding, we selected the libraries for variants with decreased cell binding in iterative rounds of binding to CHO cells. Characterization of clones from the selected libraries yielded >15 novel variants of both AAV5 and AAV6 that exhibit decreased affinity for CHO cells. Furthermore, several of these variants demonstrate altered gene delivery efficiencies to mutant CHO cell lines lacking sialic acid, suggesting some of these residues are necessary for viral binding to sialic acid and other cellular receptors. Mutation of newly identified, key receptor binding residues will remove natural receptor binding of AAV vectors and greatly enhance targeting, particularly in combination with existing rational design and directed evolution approaches. This work demonstrates that our novel forward genetics platform is an efficient and effective approach to map functional regions of the AAV capsid, further our knowledge S287