810 Oncolytic Measles Virus Induced Neutrophil Mediated Cytotoxicity Includes Antibody Dependent Cellular Cytotoxicty (ADCC) Like Activity Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright[.]
RNA VIRUS VECTORS III 808 Nonintegrating Lentiviral Vectors for Treatment of Haemophilia B Natalie J Ward,1 Luis Apolonia,1 Melanie Cochrane,2 Suzanne M Buckley,3 Simon N Waddington,3 Amit C Nathwani,2 Nicola J Philpott,1 Christine Kinnon,1 Adrian J Thrasher.1 Molecular Immunology Unit, Institute of Child Health, London, United Kingdom; 2Department of Haematology, UCL Cancer Institute, London, United Kingdom; 3Department of Haematology, Royal Free and University College Medical School, London, United Kingdom Integration deficient lentiviral vectors offer marked advantages over currently used integrating vectors as side effects caused by insertional mutagenesis are minimized Previous work has shown that efficient and sustained transgene expression in nondividing cells, such as rodent ocular, brain and muscle tissue, using integration deficient vectors can be achieved Haemophilia B is an X-linked recessive disorder caused by defects in the gene encoding coagulation factor IX Integrating lentiviral vectors have previously been used to mediate long term expression of human factor IX in vivo In this study we aim to investigate the use of nonintegrating lentiviral vectors as treatment for Haemophilia B with liver cells, primarily nondividing hepatocytes, as a principal target Integrating and nonintegrating HIV-1 based vectors expressing GFP were generated pseudotyped with glycoproteins from both Vesicular Stomatitis Virus (vsv) and Baculovirus (gp64) Vectors were first tested on differentiated and undifferentiated HuH7 human hepatocytes where transgene expression in dividing cells was lost over time due to cell division for non-integrating virus compared with stable expression for integrating vector Integrating and non-integrating VSVg pseudotyped vectors for delivery of human factor IX (hFIX) cDNA were then assessed for duration of expression in vivo in adult C57/BL6 mice After intravenous injection stable hFIX expression was detected from integrating vectors at 1500ng/mL (30% normal), however no expression was identified from nonintegrating vectors As assayed by quantitative PCR, mice injected with nonintegrating vector had significantly fewer viral genomes in liver compared with integrating vector To further investigate whether nonintegrating vectors could mediate expression of a transgene in liver vectors expressing a luciferase transgene were assessed in adult SCID mice Overall, expression from the nonintegrating vector was stable and sustained in liver but luciferase levels were significantly lower than seen with the integrating vector 809 Viral Vector Adherence to Transduced Cells Surface Depends on Vector Preparation and Is Inactivated by Human Serum Complement Martina Cesani,1 Tiziana Plati,1 Giuliana Vallanti,2 Lucia Sergi Sergi,1 Marina Radrizzani,2 Luigi Naldini,1,3 Alessandra Biffi.1 Telethon Institute for Gene Therapy (TIGET-HSR), Milano, Italy; 2MolMed S.p.A., Milano, Italy; 3Università Vita-Salute San Raffaele, Milano, Italy Prolonged adherence of viral particles to ex-vivo transduced cells is an event that has been described to cause inadvertent secondary transduction both in vitro and possibly in vivo and that raises concerns regarding the safety of utilizing viral vectors in clinical applications In the context of safety assessment of a lentiviral-based gene therapy strategy for the treatment of Metachromatic Leukodystrophy, we have performed in vitro studies aimed at assessing the extent of vector shedding by human hematopoietic stem and progenitor cells (HSPC) upon transduction Co-culture experiments of cord-blood derived CD34+ HSPC with 293T cells show that secondary transduction by cell-adherent vectors requires cell-cell contact and decreases with decreasing ratios between CD34+ and 293T cells The binding of the vector to the cell surface is stable and cannot be dissociated by S310 prolonged washes nor by post-transduction incubation with heparin, that has been demonstrated to compete with cell surface heparan sulfate for the interaction with the vector and thus to decrease transduction Human serum complement is able to inactivate the adherent vector particles, as a short in vitro wash of CD34+ transduced cells with human serum results in a 100-fold decrease of secondary transduction of 293T cells These data were confirmed using a more physiological secondary target, such as bone-marrow resident human mesenchymal stem cells Interestingly, transduction of CD34+ cells with lentiviral vector produced by a large-scale, GMP-ready purification process developed for the clinical trial gave a 100-fold reduction in the secondary transduction of 293T cells as compared to the lab-scale vectors This observation leads to the hypothesis that the binding between vector and cell is mediated by some component or condition that is eliminated in the large-scale vector purification process Taken together, these data show that in vivo secondary transduction of host cells by CD34+ cell-adherent vector particles is contrasted by clinical-grade vector preparation and serum inactivation, and thus strongly alleviate concerns regarding in vivo shedding of infectious vector particles by transplanted transduced cells 810 Oncolytic Measles Virus-Induced Neutrophil-Mediated Cytotoxicity Includes Antibody Dependent Cellular Cytotoxicty (ADCC)Like Activity Yu Zhang,1 Jennifer Thomson,1 Adele K Fielding.1 Academic Haematology, Royal Free & University College Medical School, London, United Kingdom The oncolytic property of live attenuated vaccine strain of measles virus (MV) has been demonstrated in numerous in vitro and in vivo studies Previous data from our group showed a very significant neutrophil infiltration in MV-treated human tumour xenografts in immunodeficient mice (Grote et al 2003 Cancer Research 63, 64636468) Similar observations of neutrophil infiltration have also been made after intravenous administration of oncolytic vesicular stomatitis virus in mice (Breitbatch et al 2007 Molecular Therapy 15 9, 16861693) We hypothesized that a neutrophil-mediated host immune response against measles virus-infected tumour cells might favour their elimination and we sought a mechanism to explain this First, we investigated whether MV stimulates neutrophils to kill cells via an Apo2L/tumour necrosis factor-related apoptosis inducing ligand (TRAIL)-mediated mechanism Neutrophils were clearly activated by MV infection, as determined by L-selectin shedding There was a mean 20-fold reduction in TRAIL mRNA expression-post-infection (n=9 healthy donors) This was accompanied by a small diminution in intracellular TRAIL protein Neither was cell-surface TRAIL up regulated By contrast, there was a significant increase in soluble TRAIL secretion after MV infection RNA and protein synthesis inhibitors (actinomycin D and cycloheximide) did not affect the amount of TRAIL secreted by neutrophils in response to MV Taken together, this suggested that de novo synthesis was not required for MV-mediated TRAIL release from neutrophils Treatment with monensin, which is a specific azurophilic granule release stimulator, induced an additive increase in soluble TRAIL release following MV infection This suggested that MV stimulates release of pre-fabricated TRAIL We then investigated the functional consequences of MV infection of neutrophils by carrying out neutrophil cytotoxicity assay against MV infected and uninfected target cells When MV infected neutrophils were co-cultured with Jurkat target cells, chromiumrelease assay showed consistent neutrophil cytotoxicity against both uninfected and MV-infected Jurkat cells When similar experiments were carried out in transwells, MV infected neutrophils show no specific ctotoxicity against Jurkat cells This indicated that direct cell-cell contact is required for MV-induced neutrophil cytotoxicity Interestingly, a blocking antibody against human TRAIL did not Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy RNA VIRUS VECTORS III abrogate the killing, but instead induced a consistent enhancement of cytotoxicity, as did the addition of human serum containing antiMV antibodies Thus it appears that neutrophil-mediated antibody dependent cellular cytotoxicity (ADCC)-like activity may play a role in MV-induced neutrophil cytotoxicity 811 A Novel Approach for Preventing Integration Matthew Bayer,1 Boris Kantor,1 Hong Ma,1 Thomas McCown,1,2 Tal Kafri.1,3 Gene Therapy Center, University of North Carolina-Chapel Hill, Chapel Hill, NC; 2Department of Psychiatry, University of North Carolina-Chapel Hill, Chapel Hill, NC; 3Department of Microbiology and Immunology, University of North CarolinaChapel Hill, Chapel Hill, NC Recent findings have underscored the danger of insertional mutagenesis posed by integrating retroviral and lentiviral vectors and, therefore, the utility of generating nonintegrating vectors Unfortunately, most nonintegrating vectors, designed with mutations to the integrase gene or att sequence, not completely prevent integration, with a potentially genotoxic background level of illegitimate integration remaining in the absence of integrase activity Furthermore, neither the mechanism mediating illegitimate integration, nor the species of vector genome (circular or linear) serving as its substrate, is known Here we describe a novel method, using a vector/packaging system deficient in reverse transcription, for generating an integration-deficient vector We found that the newly derived HIV-1 vector’s genomes were composed mainly of circular single-LTR species, and that Southern-blot analysis failed to detect traditional linear HIV genomes considered to be the substrate for integration Further analysis of vector genomes, using a shuttlevector assay, which allows clonal isolation of episomal circular forms, confirmed that the new vector produced 1-LTR circular genomes almost exclusively Sequence analysis of these genomes implies that inefficient strand displacement in the course of reverse transcription contributes to aberrant episome formation and reduced integration, although further investigation into the sequences of aberrantly formed vector genomes is called for Furthermore, qPCR analysis of vectortransduced cells suggests that the mutation, in combination with the commonly used D64E integrase mutation, results in an illegitimate integration level nearly three times lower than that produced by the integrase mutation alone, suggesting that, although linear vector genomes are the preferred substrate for illegitimate integration, the possibility that circular episomes contribute to this unwanted outcome cannot be ruled out Also, we found that the novel vector’s titers were comparable to those of conventional nonintegrating vectors, as were its transgene expression levels, both in cultured cells and in the rat brain Finally, because circular episomes are the predominant species produced in this novel vector system, we are evaluating the use of this system in mediating site-specific integration Overall, we believe that this new system will provide a means of reducing illegitimate integration and enhancing vector safety for future applications 812 SB Transposase-Directed Genomic Insertion of Lentiviral DNA Substrates: Novel Hybrid Lentiviral Vectors with a Random Integration Profile Brian Moldt,1 Nicklas H Staunstrup,1 Palle Villesen,2 Maria Jakobsen,1 Lajos Mátés,3 Zoltán Ivics,3 Zsuzsanna Izsvák,3 Jacob G Mikkelsen.1 Department of Human Genetics, University of Aarhus, Aarhus, Denmark; 2Bioinformatics Research Center, University of Aarhus, Aarhus, Denmark; 3Max Delbrück Center for Molecular Medicine, Berlin, Germany Integration of HIV-1-based lentiviral vector (LV) DNA is strongly biased towards transcriptionally active loci resulting in an increased risk of insertional mutagenesis and transformation In an attempt to bypass the natural lentiviral integration pathway, we present here a novel hybrid vector technology based on Sleeping Beauty (SB) transposase-mediated insertion of viral DNA delivered by integrase-defective lentiviral vectors (IDLVs) We constructed an LV/SB hybrid vector containing a puroR gene as well as the left and right inverted repeats of SB in a context allowing transposition exclusively from circular DNA substrates Co-transduction of 293 cells with the transposon substrate vector and IDLVs encoding the hyperactive SB100X transposase resulted in a 13-fold increase in the number of drug-resistant colonies compared to background To investigate the integration properties of the LV/SB vector a total of 87 integration sites were mapped Notably, 70 % of these sites were mapped to regions outside transcriptional units, whereas the remaining 30 % were mapped to intragenic regions, demonstrating a random integration profile of the hybrid vector Using an optimized LV/ SB vector design, we carried out co-transduction studies in Chang, HepG2, HT-1080, HeLa, and 293 cells In all cell lines, we observed a robust transposase-dependent increase (up to 23-fold) in viral titers By creating an LV/SB vector containing SB-T2 inverted repeats, the viral titer was increased 42 times above background in 293 cells In primary human keratinocytes high levels of transposition from viral substrates resulted in viral titers that were ∼160-fold above background In summary, we provide proof-of-principle that lentiviral DNA can act as substrate for SB transposase-directed gene insertion in primary human cells Importantly, our data suggest that the biological constraints, leading to gene-targeted insertion by the normal lentiviral integration machinery, are resolved by the transposase resulting in a random lentiviral integration profile We therefore believe that such hybrid vectors may become valuable tools for modifying the gene integration profile of lentiviral vectors in gene therapy 813 Quantitative Analysis of ProliferationControlled Gene Transfer Efficiency and Its Implications in In Vivo Gene Delivery Charles Xue,1 Seong Sil Chang,1 Young Jik Kwon.1 Biomedical Eng, Pharmaceutical Sci, and Chemical Eng & Materials Sci, University of Calfornia, Irvine, CA Cellular proliferation is a major determining factor in gene delivery It is well-known that gene delivery to actively dividing cells is more efficient than quiescent cells For example, retroviruses can transduce only actively dividing cells and that is a reason for their limited use in gene delivery in vivo where few cells rapidly proliferate Quantitative gene transfer efficiency of gene carriers, including viral and nonviral vectors, to the cells under different proliferation conditions in one system (like in the body) has not been fully elucidated Usually gene transfer efficiency cannot be quantified until cells express transgene; therefore, post-transfection (or transduction) incubation is required and the real gene transfer efficiency at the time of gene transfer is hard to determine Significant limitations of these methods in accurately evaluating vector’s capability are: 1) It is not feasible to differentiate Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy S311 ... antibodies Thus it appears that neutrophil- mediated antibody dependent cellular cytotoxicity (ADCC)- like activity may play a role in MV -induced neutrophil cytotoxicity 811 A Novel Approach for Preventing...RNA VIRUS VECTORS III abrogate the killing, but instead induced a consistent enhancement of cytotoxicity, as did the addition of human serum containing antiMV antibodies Thus it appears that neutrophil- mediated. .. Cellular proliferation is a major determining factor in gene delivery It is well-known that gene delivery to actively dividing cells is more efficient than quiescent cells For example, retroviruses