Luciferase Marker Gene Research Articles

Correlating IgG Levels with Neutralising Antibody Levels to Indicate Clinical Protection in Healthcare Workers at Risk during a Measles Outbreak.

The rapid transmission of measles poses a great challenge for measles elimination. Thus, rapid testing is required to screen the health status in the population during measles outbreaks. A pseudotype-based virus neutralisation assay was used to measure neutralising antibody titres in serum samples collected from healthcare workers in Sheffield during the measles outbreak in 2016. Vesicular stomatitis virus (VSV) pseudotypes bearing the haemagglutinin and fusion glycoproteins of measles virus (MeV) and carrying a luciferase marker gene were prepared; the neutralising antibody titre was defined as the dilution resulting in 90% reduction in luciferase activity. Spearman’s correlation coefficients between IgG titres and neutralising antibody levels ranged from 0.40 to 0.55 (p < 0.05) or from 0.71 to 0.79 (p < 0.0001) when the IgG titres were obtained using different testing kits. In addition, the currently used vaccine was observed to cross-neutralise most circulating MeV genotypes. However, the percentage of individuals being “well-protected” was lower than 95%, the target rate of vaccination coverage to eliminate measles. These results demonstrate that the level of clinical protection against measles in individuals could be inferred by IgG titre, as long as a precise correlation has been established between IgG testing and neutralisation assay; moreover, maintaining a high vaccination coverage rate is still necessary for measles elimination.

A Unique Gene-Silencing Approach, Using an Intelligent RNA Expression Device (iRed), Results in Minimal Immune Stimulation When Given by Local Intrapleural Injection in Malignant Pleural Mesothelioma.

Background: We have recently introduced an intelligent RNA expression device (iRed), comprising the minimum essential components needed to transcribe short hairpin RNA (shRNA) in cells. Use of iRed efficiently produced shRNA molecules after transfection into cells and alleviated the innate immune stimulation following intravenous injection. Methods: To study the usefulness of iRed for local injection, the engineered iRed encoding luciferase shRNA (Luc iRed), complexed with cationic liposomes (Luc iRed/liposome-complexes), was intrapleurally injected into an orthotopic mesothelioma mouse model. Results: Luc iRed/liposome-complexes markedly suppressed the expression of a luciferase marker gene in pleurally disseminated mesothelioma cells. The suppressive efficiency was correlated with the expression level of shRNA within the mesothelioma cells. In addition, intrapleural injection of iRed/liposome-complexes did not induce IL-6 production in the pleural space and consequently in the blood compartment, although plasmid DNA (pDNA) or dsDNA (the natural construct for iRed) in the formulation did. Conclusion: Local delivery of iRed could augment the in vivo gene silencing effect without eliciting pronounced innate immune stimulation. Our results might hold promise for widespread utilization of iRed as an RNAi-based therapeutic for intracelial malignant cancers.

Genomic integration of the full-length dystrophin coding sequence in Duchenne muscular dystrophy induced pluripotent stem cells.

The plasmid vectors that express the full-length human dystrophin coding sequence in human cells was developed. Dystrophin, the protein mutated in Duchenne muscular dystrophy, is extraordinarily large, providing challenges for cloning and plasmid production in Escherichia coli. The authors expressed dystrophin from the strong, widely expressed CAG promoter, along with co-transcribed luciferase and mCherry marker genes useful for tracking plasmid expression. Introns were added at the 3' and 5' ends of the dystrophin sequence to prevent translation in E. coli, resulting in improved plasmid yield. Stability and yield were further improved by employing a lower-copy number plasmid origin of replication. The dystrophin plasmids also carried an attB site recognized by phage phiC31 integrase, enabling the plasmids to be integrated into the human genome at preferred locations by phiC31 integrase. The authors demonstrated single-copy integration of plasmid DNA into the genome and production of human dystrophin in the human 293 cell line, as well as in induced pluripotent stem cells derived from a patient with Duchenne muscular dystrophy. Plasmid-mediated dystrophin expression was also demonstrated in mouse muscle. The dystrophin expression plasmids described here will be useful in cell and gene therapy studies aimed at ameliorating Duchenne muscular dystrophy.

Enhanced immunosurveillance for animal morbilliviruses using vesicular stomatitis virus (VSV) pseudotypes

The measurement of virus-specific neutralising antibodies represents the “gold-standard” for diagnostic serology. For animal morbilliviruses, such as peste des petits ruminants (PPRV) or rinderpest virus (RPV), live virus-based neutralisation tests require high-level biocontainment to prevent the accidental escape of the infectious agents. In this study, we describe the adaptation of a replication-defective vesicular stomatitis virus (VSVΔG) based pseudotyping system for the measurement of neutralising antibodies against animal morbilliviruses. By expressing the haemagglutinin (H) and fusion (F) proteins of PPRV on VSVΔG pseudotypes bearing a luciferase marker gene, neutralising antibody titres could be measured rapidly and with high sensitivity. Serological responses against the four distinct lineages of PPRV could be measured simultaneously and cross-neutralising responses against other morbilliviruses compared. Using this approach, we observed that titres of neutralising antibodies induced by vaccination with live attenuated PPRV were lower than those induced by wild type virus infection and the level of cross-lineage neutralisation varied between vaccinates. By comparing neutralising responses from animals infected with either PPRV or RPV, we found that responses were highest against the homologous virus, indicating that retrospective analyses of serum samples could be used to confirm the nature of the original pathogen to which an animal had been exposed. Accordingly, when screening sera from domestic livestock and wild ruminants in Tanzania, we detected evidence of cross-species infection with PPRV, canine distemper virus (CDV) and a RPV-related bovine morbillivirus, suggesting that exposure to animal morbilliviruses may be more widespread than indicated previously using existing diagnostic techniques.

Efficient generation of vesicular stomatitis virus (VSV)-pseudotypes bearing morbilliviral glycoproteins and their use in quantifying virus neutralising antibodies

Morbillivirus neutralising antibodies are traditionally measured using either plaque reduction neutralisation tests (PRNTs) or live virus microneutralisation tests (micro-NTs). While both test formats provide a reliable assessment of the strength and specificity of the humoral response, they are restricted by the limited number of viral strains that can be studied and often present significant biological safety concerns to the operator. In this study, we describe the adaptation of a replication-defective vesicular stomatitis virus (VSVΔG) based pseudotyping system for the measurement of morbillivirus neutralising antibodies. By expressing the haemagglutinin (H) and fusion (F) proteins of canine distemper virus (CDV) on VSVΔG pseudotypes bearing a luciferase marker gene, neutralising antibody titres could be measured rapidly and with high sensitivity. Further, by exchanging the glycoprotein expression construct, responses against distinct viral strains or species may be measured. Using this technique, we demonstrate cross neutralisation between CDV and peste des petits ruminants virus (PPRV). As an example of the value of the technique, we demonstrate that UK dogs vary in the breadth of immunity induced by CDV vaccination; in some dogs the neutralising response is CDV-specific while, in others, the neutralising response extends to the ruminant morbillivirus PPRV. This technique will facilitate a comprehensive comparison of cross-neutralisation to be conducted across the morbilliviruses.

Anti-tumour activity of oncolytic Western Reserve vaccinia viruses in canine tumour cell lines, xenografts, and fresh tumour biopsies.

Cancer is one of the most common reasons for death in dogs. One promising approach is oncolytic virotherapy. We assessed the oncolytic effect of genetically modified vaccinia viruses in canine cancer cells, in freshly excised tumour biopsies, and in mice harbouring canine tumour xenografts. Tumour transduction efficacy was assessed using virus expressing luciferase or fluorescent marker genes and oncolysis was quantified by a colorimetric cell viability assay. Oncolytic efficacy in vivo was evaluated in a nude mouse xenograft model. Vaccinia virus was shown to infect most tested canine cancer cell lines and primary surgical tumour tissues. Virus infection significantly reduced tumour growth in the xenograft model. Oncolytic vaccinia virus has antitumour effects against canine cancer cells and experimental tumours and is able to replicate in freshly excised patient tumour tissue. Our results suggest that oncolytic vaccinia virus may offer an effective treatment option for otherwise incurable canine tumours.

British Society for Gene and Cell Therapy (BSGCT) Conference AbstractsRoyal HollowayUniversity of London17–19 April, 2013

British Society for Gene and Cell Therapy (BSGCT) Conference AbstractsRoyal HollowayUniversity of London17–19 April, 2013

Few genes are associated with the capability of pancreatic ductal adenocarcinoma cells to grow in the liver of nude rats

Owing to aggressiveness and chemoresistance, pancreatic ductal adenocarcinoma (PDAC) is characterised by a poor prognosis. To address this disease-spe-cific dilemma we aimed to establish animal models, which can be used for identifying new specific tumor markers, as well as serving as tools for potential therapeutic approaches. From a panel of sixteen pancreatic cancer cell lines, two human (Suit2-007 and Suit2-013) and a rat (ASML) cell line were selected for their properties to grow in the liver of male RNU rats and mimic liver metastasis of PDAC. For better monitoring of metastatic tumor growth invivo, all three pancreatic cancer cell lines were stably transfected with eGFP and luciferase marker genes. In addition, the mRNA expression profile of 13 human PDAC cell lines was analyzed by BeadChip array analysis. Only 33 genes and 5 signaling pathways were identified as significantly associated with the ability of the cell lines to grow initially and/or consistently in rat liver. Only a minority of these genes (osteopontin, matrix metalloproteinase-1 and insulin-like growth factor 1) has been intensively studied and shown to be closely related to cancer progression. The function of the remaining 30 genes ranges from moderate to poorly investigated, and their function in cancer progression is still unclear. The ensuing three pancreatic cancer liver metastasis models vary in their aggressiveness and macroscopic growth. They will be used for preclinical evaluation of new therapeutic approaches aiming at the genes identified.

Inhibitors of Alphavirus Entry and Replication Identified with a Stable Chikungunya Replicon Cell Line and Virus-Based Assays

Chikungunya virus (CHIKV), an alphavirus, has recently caused epidemic outbreaks and is therefore considered a re-emerging pathogen for which no effective treatment is available. In this study, a CHIKV replicon containing the virus replicase proteins together with puromycin acetyltransferase, EGFP and Renilla luciferase marker genes was constructed. The replicon was transfected into BHK cells to yield a stable cell line. A non-cytopathic phenotype was achieved by a Pro718 to Gly substitution and a five amino acid insertion within non-structural protein 2 (nsP2), obtained through selection for stable growth. Characterization of the replicon cell line by Northern blotting analysis revealed reduced levels of viral RNA synthesis. The CHIKV replicon cell line was validated for antiviral screening in 96-well format and used for a focused screen of 356 compounds (natural compounds and clinically approved drugs). The 5,7-dihydroxyflavones apigenin, chrysin, naringenin and silybin were found to suppress activities of EGFP and Rluc marker genes expressed by the CHIKV replicon. In a concomitant screen against Semliki Forest virus (SFV), their anti-alphaviral activity was confirmed and several additional inhibitors of SFV with IC50 values between 0.4 and 24 µM were identified. Chlorpromazine and five other compounds with a 10H-phenothiazinyl structure were shown to inhibit SFV entry using a novel entry assay based on a temperature-sensitive SFV mutant. These compounds also reduced SFV and Sindbis virus-induced cytopathic effect and inhibited SFV virion production in virus yield experiments. Finally, antiviral effects of selected compounds were confirmed using infectious CHIKV. In summary, the presented approach for discovering alphaviral inhibitors enabled us to identify potential lead structures for the development of alphavirus entry and replication phase inhibitors as well as demonstrated the usefulness of CHIKV replicon and SFV as biosafe surrogate models for anti-CHIKV screening.

Targeting of prostate cancer cells by a cytotoxic lentiviral vector containing a prostate stem cell antigen (PSCA) promoter

The efficacy of prostate cancer gene therapy is limited by the inefficiency of prostate-specific promoters as compared to ubiquitous viral promoters. The purpose of this investigation was to evaluate the specificity and efficacy of a lentiviral vector driven by a PSCA promoter. Prostate cancer (LNCap, C42-B, and LAPC-4) and non-prostate cancer (HeLa, MB231, and MCF-7) cells were transduced with a lentiviral vector expressing either the luciferase or the HSV-TK suicide gene and driven by a short PSCA promoter. Specificity and efficacy were evaluated in vitro and in vivo. Luciferase expression was only detected in prostate cancer cells and was comparable to the universal CMV promoter. Luciferase expression in prostate cancer cells cultured with androgen was higher than that in cells cultured without androgen. In subsequent cytotoxicity experiments in which the luciferase marker gene was replaced with the HSV-TK gene, the lentiviral vector harboring the PSCA promoter induced cytotoxicity in prostate cancer cell lines while demonstrating a minimal effect on non-prostate cells. Cellular toxicity was correlated to increasing concentrations of the prodrug ganciclovir. Androgen had a positive effect on the cytotoxicity of this lentiviral construct. Intratumoral injection of prostate cancer xenografts with the lentiviral construct induced tumor growth inhibition versus saline controls. Our results indicate that a lentiviral gene therapy vector driven by a short PSCA promoter can induce prostate-specific cellular toxicity in vivo and in vitro and may provide a strategy to selectively treat local and advanced metastatic prostate cancer. Prostate 69: 1422-1434, 2009. (c) 2009 Wiley-Liss, Inc.

Long-term transgene expression in mouse neural progenitor cells modified with ϕC31 integrase

Stem cells can potentially be utilized in combined gene/cell therapies for neural diseases. We examined the ability of the non-viral ϕC31 integrase system to promote stable transgene expression in mouse neural progenitor cells (mNPCs). ϕC31 integrase catalyzes the sequence-specific integration of attB-containing plasmids into pseudo attP sites in mammalian genomes, to produce long-term transgene expression. We achieved gene transfer by co-nucleofection of a plasmid carrying the luciferase marker gene and an attB site and a plasmid expressing integrase in mNPCs that had been generated in a neurosphere preparation. Luciferase expression was quantified in live cells for 8 weeks, revealing persistence of gene expression. Sequence-specific integration at a preferred pseudo attP site in the mouse genome was detected by using PCR. Furthermore, sustained transgene expression was demonstrated in genetically modified NPCs that were cultured in conditions that promoted either growth or differentiation into neurons and astrocytes. Our results demonstrate that the ϕC31 integrase system produces stable transgene expression in adult mNPCs and their progeny and may be useful in strategies for combating neurodegenerative disorders.

Mammalian Luciferase Detection Vectors and Applications

The light‐emitting reaction of the marine bioluminescent bacterium Vibrio harveyi is catalyzed by the bacterial luciferase enzyme which exists as an alpha‐beta heterodimer encoded by the luxA and luxB genes with subunit molecular weights of 42K and 37K respectively. The enzyme catalyzes a reaction with FMNH2, oxygen and a long‐chain aldehyde as substrates to yield visible light at 490 nm. A new luciferase marker gene detection system has been developed based upon this bacterial luciferase isolated from Vibrio harveyi. Sequences encoding the two luciferase subunits, luxA and luxB have been cloned into two separate vectors. These vectors also include a CMV promoter for expression in mammalian cells as well as an ampicillin resistance gene (amp) for selection and amplification, the SV40 polyadenylation sequence and the SD/SA‐RNA splice donor and acceptor sequence for maximum expression. The vectors were tested for light emission parameters in a live mammalian cell format and with cell lysate samples from NIH3T3 murine fibroblasts in conjunction with transfection using vector GC:11726 Mus musculus biliverdin reductase B (flavin reductase (NADPH)) sequence for production of the reduced flavin mononucleotide (FMNH2) cofactor intracellularly. These systems are being developed to monitor regulation of expression for two independent vector constructs, upon the dual expression.

A simple, lanthanide-based method to enhance the transduction efficiency of adenovirus vectors

Based upon the powerful bridging and charge-masking properties of lanthanide cations (Ln3+), we have investigated their use to improve the transduction efficiency of adenovirus vectors. Using a luciferase marker gene, it was possible to increase transgene expression by the murine mesenchymal stem cell line C3H10T(1/2) by up to four log orders when using very low multiplicities of infection in conjunction with Ln3+; La3+ was superior to Gd3+, Y3+ and Lu3+ in this regard. All Ln3+ were more effective than Ca2+. Flow cytometry, using a green fluorescent protein marker gene, confirmed that La3+ increased both the percentage of transduced cells and the level of transgene expression per cell. Transduction of primary cultures of a variety of different mesenchymal cells from human, rabbit, bovine and rat sources, as well as gene transfer to synovium and muscle in vivo, was also greatly enhanced. Our findings suggest that this lanthanide-based method holds much promise for expediting both experimental and clinical applications of gene transfer with adenoviral vectors.

Generation of a novel replication-incompetent adenoviral vector derived from human adenovirus type 49: manufacture on PER.C6 cells, tropism and immunogenicity

Recombinant adenoviral vectors based on type 5 (rAd5) show great promise as a vaccine carrier. However, neutralizing activity against Ad5 is prevalent and high-titred among human populations, and significantly dampens Ad5-based vaccine modalities. The generation of alternative adenoviral vectors with low seroprevalence thus receives much research attention. Here, it is shown that a member from human adenovirus subgroup D, i.e. Ad49, does not cross-react with Ad5 neutralizing activity, making it a candidate serotype for vector development. Therefore, a plasmid system that allows formation of replication-incompetent adenovirus serotype 49 vaccine vectors (rAd49) was constructed and it was demonstrated that rAd49 can be successfully propagated to high titres on existing Ad5.E1-complementing cell lines such as PER.C6. Using an rAd49 vector carrying the luciferase marker gene, detailed seroprevalence studies were performed, demonstrating that rAd49 has low seroprevalence and neutralizing antibody titres worldwide. Also, we have initiated rAd49 vector receptor usage suggesting that rAd49 utilizes hCD46 as a cellular receptor. Finally, the immunogenicity of the rAd49 vector was assessed and it was shown that an rAd49.SIVGag vaccine induces strong anti-SIVGag CD8+ T-lymphocytes in naïve mice, albeit less than an rAd5.SIVGag vaccine. However, in mice with high anti-Ad5 immunity the rAd5.SIVGag vaccine was severely blunted, whereas the anti-SIVGag response was not significantly suppressed using the rAd49.SIVGag vaccine. These data demonstrate the potential of a replication deficient human group D adenoviral vector for vaccination purposes.

450. Novel, Injectable, Genetically Engineered Stem Cell-Based System for Anterior Spinal Fusion

Spinal fusion has become a popular surgical technique used to provide segmental fixation and to maintain stability and correct deformity of the spine. Stem cell|[ndash]|based therapy involving genetically engineered mesenchymal stem cells (MSCs) and bone morphogenetic protein (BMP) genes can promote bone formation and lead to spinal fusion. Exogenous control and monitoring of transgene expression in vivo is of great importance for gene therapy. In such a system, transgene expression can be regulated over time at a defined level of expression, thus controlling the regeneration process. We hypothesized that direct injection of MSCs that overexpress the human BMP-2 (hBMP-2) gene under a tetracycline-controlled expression system (Tet-Off system) could induce regulated bone formation and lead to interbody anterior spinal fusion. We used a Tet-Off system in which genetically engineered MSCs harbor the inducible human BMP-2 expression vector. The system was regulated by the administration of Doxycycline (Dox), a tetracycline analog. Nude rats received an injection of 10^6 genetically engineered MSCs into two caudal intervertebral discs under fluoroscopic guidance. In the negative control group, the rats were supplied with Dox in their drinking water. A quantitative analysis of vertebra anterior fusion was performed using micro-computerized tomography (|[mu]|CT) scanning. In addition, engineered MSCs were also infected with retroviruses encoding for the luciferase or green fluorescent protein (GFP) marker genes. In vivo cell survival was monitored noninvasively and quantitatively by using a bioluminescence imaging system and a novel in vivo fibered confocal microscopy system (Cell Vizio). Quantitative |[mu]|CT analyses demonstrate extensive bone formation in the disc region, leading to interbody fusion (Figure 1). We did not find any new bone formation in the control animals, in which hBMP-2 expression had been inhibited. Longitudinal monitoring of the luciferase and GFP-labeled MSCs in vivo revealed a high bioluminescence signal (Figure 2A, arrows). GFP expressing cells were detected in the intervertebral disc with Cell-Vizio up to 4 weeks postinjection (Figure 2B). Our data demonstrate for the first time the use of an injectable, regulated, and monitored system for the induction of anterior spinal fusion. These results may pave the way toward the advancement of a biological solution for spinal fusion that will replace currently nonoptimal prosthetic implants.

Liposome Vector Containing Biosurfactant-Complexed DNA as Herpes Simplex Virus Thymidine Kinase Gene Delivery System

For injectable-sized liposome complexed with DNA (lipoplexes) with high transfection efficiency of genes, we initially prepared small-sized liposomes by addition of biosurfactant. For selectivity of gene expression, the thymidine kinase (MK-tk) gene controlled by midkine was used for herpes simplex virus thymidine kinase (HSV-tk) gene therapy. Liposomes composed of 3([N-(N′,N′–dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol), L-dioleoylphosphatidylethanolamine (DOPE), and a biosurfactant, such as β-sitosterol β-D-glucoside (Sit-G) for Sit-G-liposomes and mannosylerythrytol lipid A (MEL) for MEL-liposomes, produced about 300-nm-sized lipoplexes. Sit-G- and MEL-liposomes showed higher transfection efficiency of the luciferase marker gene and thymidine kinase activity in the presence of serum in the cells. The treatment with transfection of MK-tk gene by Sit-G-liposome and injection of ganciclovir significantly reduced tumor growth in a solid tumor model, compared with that by Sit-G-liposome alone. This finding suggested that Sit-G-liposome is a potential vector for HSV-tk gene therapy.

954. High Intensity Focused Ultrasound (HIFU) Induced Trans-Gene Activation in Solid Tumors

This work investigated the feasibility of HIFU-induced gene activation in sublethally injured cancer cells in a murine tumor model. 4T1 mouse mammary cancer cells were implanted subcutaneously in the hindlimbs of Balb/C mice and luciferase marker gene vectors (Ad-hsp-Luc) under the control of a heat-sensitive promoter (hsp70B) were injected intratumorly (2x108 pfu/tumor) for dissemination and gene transfection. One day following virus injection, targeted tumors (D=6 8mm) were heated to a peak temperature of 550C, 650C, 750C and 850C (measured with thermocouples), respectively, for 5s-20s at multiple sites by using a focused 1.1-MHz HIFU transducer. The time course of HIFU- induced luciferase gene expression was monitored and quantified using a Xenogen in vivo bioluminescence imaging system for one week. Inducible luciferase gene expression was found to be highly thermal dose dependent with total luminescence intensity increased by 15 – 120 fold at different HIFU exposure conditions compared to the control group. The maximum gene activation (120 fold) was induced at 650C-10s and 750C-10s one day after HIFU exposure and decayed gradually within 7 days. Histology examinations (H&E staining) of the tumor tissue one day after 750C-10s HIFU exposure revealed multiple focal areas of necrotic injury surrounded by sublethally injured and unaffected tumor tissues. Corresponding tumor growth after 750C-10s HIFU exposure was reduced by 50% from the original volume within 20 days while the tumor volume in the control group increased by 3 fold. Altogether, these results demonstrated the feasibility of HIFU-induced transgene activation presumably in sublethally injured tumor tissues. This observation opens up the possibility for the combination of HIFU-induced thermal ablation with simultaneous heat-induced gene therapy for cancer therapy. Furthermore, efforts are also underway to explore HIFU-targeted gene delivery to solid tumors. This work investigated the feasibility of HIFU-induced gene activation in sublethally injured cancer cells in a murine tumor model. 4T1 mouse mammary cancer cells were implanted subcutaneously in the hindlimbs of Balb/C mice and luciferase marker gene vectors (Ad-hsp-Luc) under the control of a heat-sensitive promoter (hsp70B) were injected intratumorly (2x108 pfu/tumor) for dissemination and gene transfection. One day following virus injection, targeted tumors (D=6 8mm) were heated to a peak temperature of 550C, 650C, 750C and 850C (measured with thermocouples), respectively, for 5s-20s at multiple sites by using a focused 1.1-MHz HIFU transducer. The time course of HIFU- induced luciferase gene expression was monitored and quantified using a Xenogen in vivo bioluminescence imaging system for one week. Inducible luciferase gene expression was found to be highly thermal dose dependent with total luminescence intensity increased by 15 – 120 fold at different HIFU exposure conditions compared to the control group. The maximum gene activation (120 fold) was induced at 650C-10s and 750C-10s one day after HIFU exposure and decayed gradually within 7 days. Histology examinations (H&E staining) of the tumor tissue one day after 750C-10s HIFU exposure revealed multiple focal areas of necrotic injury surrounded by sublethally injured and unaffected tumor tissues. Corresponding tumor growth after 750C-10s HIFU exposure was reduced by 50% from the original volume within 20 days while the tumor volume in the control group increased by 3 fold. Altogether, these results demonstrated the feasibility of HIFU-induced transgene activation presumably in sublethally injured tumor tissues. This observation opens up the possibility for the combination of HIFU-induced thermal ablation with simultaneous heat-induced gene therapy for cancer therapy. Furthermore, efforts are also underway to explore HIFU-targeted gene delivery to solid tumors.

596. A Novel Replication Incompetent Human Subgroup D Adenoviral Vector Based on Type 49: Manufacture on PER.C6®, Cell Tropism and Immunogenicity

Adenoviral Recombinant vectors based on type 5 (rAd5) show great promise as vaccine carrier. However, neutralizing activity against Ad5 is prevalent and high titered among human populations and significantly dampens Ad5-based vaccine modalities. The generation of alternative adenoviral vectors with low sero-prevalence thus receives much research interest. We have previously published that several members of human subgroup D adenoviruses display low sero-prevalence in Europe and here we demonstrate that one such member, i.e. Ad49, is immunologically distinct from Ad5. We next set out to construct a plasmid system that allows formation of replication incompetent adenovirus serotype 49 vaccine vectors (rAd49) and demonstrate that rAd49 can be successfully propagated to high titers on existing E1-complementing cell lines such as PER.C6®. Using a rAd49 vector carrying the luciferase marker gene sero-prevalence studies were performed demonstrating that rAd49 has low sero-prevalence and neutralizing antibody titers worldwide. Also, we have initiated rAd49 vector receptor usage suggesting that rAd49 utilizes hCD46 as a cellular receptor. Next, we assessed the immunogenicity of the rAd49 vector and show that a rAd49.SIVGag vaccine induces strong anti-SIVGag CD8+T-lymphocytes in naive mice albeit approximately 1.5-fold less compared to a rAd5.SIVGag vaccine. However, in mice with high anti-Ad5 immunity the anti- SIVGag response was not statistically significant suppressed using the rAd49.SIVGag vaccine whereas the rAd5.SIVGag vaccine was severely blunted. Finally, rAd35.SIVGag prime-rAd49.SIVGag boost experiments in mice carrying high-level anti-Ad5 neutralizing activity demonstrated that the rAd49 based vaccine is not hampered by neither rAd5 nor rAd35 neutralizing activity. These data demonstrate the potential of a replication deficient human D-group adenoviral vector as stand-alone vaccine carrier or in prime-boost settings with other rare human adenoviral vectors.

A Novel In Vivo Animal Model for Human Multiple Myeloma Based on Bioluminescence Imaging of Tumor Cell Growth.

Preclinical testing of new therapeutical strategies for the treatment of multiple myeloma (MM) requires animal models that closely resemble human disease and allow quantitative evaluation of the applied therapy. Models that meet both requirements have thus far not been described. Here we present a novel in vivo MM model by engraftment of MM U266 or RPMI-8226/S cells, both of human origin, into RAG2γc double knock-out mice. These mice are totally immune deficient because they lack T-, B and NK cells and the mice easily accept human cells (van Rijn et al., Blood 2003, Rozemuller et al., 2004). After intravenous injection of 2x106 MM cells engraftment and outgrowth occurred in all mice but was limited to the bone marrow compartment only. Flow cytometry (FCM) confirmed the presence of human CD45/38/138 positive MM cells in femur, spine, tibia and sternum bone specimens. Infiltration into other organs was not observed. In a next step MM cells were stably transduced using a retroviral vector encoding both the Green Fluorescent Protein (GFP) and firefly Luciferase (fLuc) marker genes. Technical advances in recent years in optical imaging by Bioluminescence Imaging (BLI) techniques allow visualization and quantification of bioluminescent light by detecting photons that are transmitted through mammalian tissue. When luciferase converts the substrate luciferin, photons are emitted that can be registered by using sensitive CCCD cameras. The absolute number of photons that are produced correlates, in our application, with local tumor mass. Mice were injected i.v. with 2x106 GFP-fLuc transduced MM cells (U266 or RPMI8226/S) and imaged weekly using BLI. Within 2 weeks after injection significant BLI signals were detectable. Per mouse 5-10 foci showed luciferase activity, predominantly in the pelvic region, skull, limbs, sternum, ribs and the spine. This low frequency of engraftment is in line with earlier reports on RPMI8226/S (Mitsiades et al., Cancer Res 2003). At 9 weeks the first mice developed hind leg paralysis which could be attributed to tumor associated spinal lesions. After 12 weeks the last mouse was sacrificed. BLI revealed that the intensity of light production at the various sites of tumor growth within individual mice as well as between mice showed a similar increase. This reflects an increase in tumor mass. Quantitative analysis of subsequent BLI images allowed construction of tumor growth curves of the total tumor mass per mouse as well as for the individual foci of MM growth in individual mice. We typically observed exponential growth, with growth curves running parallel with an average population doubling time of approximately 4–5 days. The range in which tumor growth could be monitored (and as a consequence also the response to treatment) spans 3–4 decades. In contrast with previously reported murine models for human MM where -next to bone marrow homing- also extra-skeletal tumors were observed our model almost exclusively shows homing of MM cells to the BM and is therefore more consistent with the clinical manifestation in myeloma patients. The major advantage of the model is the option for quantitative evaluation of the effect of a given treatment on the tumorload. Currently we are studying the efficacy of newly developed geranyl-geranyl-transferase inhibitors (GGTI).In conclusion, we have developed a novel in vivo model to study the characteristics of homing and outgrowth of MM and for quantitative evaluation of the efficacy of the therapeutic intervention applied.

1103. Site-Specific Chromosomal Integration in Human Mesenchymal Stem Cells Mediated by PhiC31 Integrase

Human mesenchymal stem cells (hMSCs) have emerged as a promising new tool in gene therapy/ cell therapy strategies due to their potential use in a wide variety of applications, including both short- and long-term regeneration. With viral vectors being frequently criticized for their immunogenicity, random integration and difficult production, alternatives are desirable. A safe and effective gene delivery method resulting in targeted chromosomal integration with prolonged therapeutic levels of gene expression would be valuable. PhiC31 integrase, a site-specific recombinase from phage phiC31, pairs an attB recognition site on an incoming plasmid with pseudo attP sites in mammalian genomes. This integrase has been used to achieve genomic integration in human keratinocytes, mouse liver, rat retina and rabbit joints. Here we describe non-viral approaches for gene delivery using phiC31 integrase, transfected either as mRNA or as a DNA expression vector, in conjugation with an attB-containing donor plasmid. We evaluated the efficacy of mRNA encoding phiC31 integrase to mediate unidirectional integration into pseudo attP sites when co-transfected with an attB donor plasmid containing a transgene of interest. We also demonstrated that co-transfection of a plasmid expressing phiC31 integrase with a plasmid containing the luciferase marker gene and an attB site resulted in 39-fold higher expression of luciferase as assessed by live imaging, compared to control transfections without integrase. Expansion of the integrants and their differentiation into the osteogenic phenotype revealed sustained gene expression, indicating that the integration sites used in undifferentiated hMSCs continued to provide gene expression after differentiation of the cells. Finally, plasmid rescue and DNA sequence analysis of the integration sites showed that the integration events occurred at several pseudo attP sites in the human genome. Summarizing our results, phiC31 integrase, provided either as mRNA or DNA, mediates site-specific integration of transgenes and can provide high levels of gene expression in hMSCs and differentiated osteogenic cells. These findings suggest that phiC31 integrase represents an efficient and practical non-viral approach for genetic modification of hMSCs that could have potential for clinical applications.