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Sample records for crgd targeting vectors

  1. Targeted adenoviral vectors

    NASA Astrophysics Data System (ADS)

    Douglas, Joanne T.

    The practical implementation of gene therapy in the clinical setting mandates gene delivery vehicles, or vectors, capable of efficient gene delivery selectively to the target disease cells. The utility of adenoviral vectors for gene therapy is restricted by their dependence on the native adenoviral primary cellular receptor for cell entry. Therefore, a number of strategies have been developed to allow CAR-independent infection of specific cell types, including the use of bispecific conjugates and genetic modifications to the adenoviral capsid proteins, in particular the fibre protein. These targeted adenoviral vectors have demonstrated efficient gene transfer in vitro , correlating with a therapeutic benefit in preclinical animal models. Such vectors are predicted to possess enhanced efficacy in human clinical studies, although anatomical barriers to their use must be circumvented.

  2. cRGD Peptide-Conjugated Icosahedral closo-B12 2− Core Carrying Multiple Gd3+-DOTA Chelates for αvβ3 Integrin-Targeted Tumor Imaging (MRI)

    PubMed Central

    Goswami, Lalit N.; Ma, Lixin; Cai, Quanyu; Sarma, Saurav J.; Jalisatgi, Satish S.; Hawthorne, M. Frederick

    2013-01-01

    A vertex-differentiated icosahedral closo-B122− core was utilized to construct a αvβ3 integrin receptor-targeted (via cRGD peptide) high payload MRI contrast agent (CA-12) carrying 11 copies of Gd3+-DOTA chelates attached to the closo-B122− surface via suitable linkers. The resulting polyfunctional MRI contrast agent possessed a higher relaxivity value per-Gd compared to Omniscan, a small molecular contrast agent commonly used in clinical settings. The αvβ3 integrin receptor specificity of CA-12 was confirmed via in vitro cellular binding experiments and in vivo MRI of mice bearing human PC-3 prostate cancer xenografts. Integrin αvβ3-positive MDA-MB-231 cells exhibited 300% higher uptake of CA-12 than αvβ3-negative T47D cells. Serial T1-weighted MRI showed superior contrast enhancement of tumors by CA-12 compared to both a non-targeted 12-fold Gd3+-DOTA closomer control (CA-7) and Omniscan. Contrast enhancement by CA-12 persisted for 4 h post-injection, and subsequent enhancement of kidney tissue indicated a renal elimination route similar to Omniscan. No toxic effects of CA-12 were apparent in any mice for up to 24 h post-injection. Post-mortem ICP-OES analysis at 24 hours detected no residual Gd in any of the tissue samples analyzed. PMID:23391150

  3. cRGD conjugated mPEG-PLGA-PLL nanoparticles for SGC-7901 gastric cancer cells-targeted Delivery of fluorouracil.

    PubMed

    Liu, Peifeng; Wang, Hongbin; Wang, Qi; Sun, Ying; Shen, Ming; Zhu, Mingjie; Wan, Zhiyong; Duan, Yourong

    2012-06-01

    The main purpose of this study was to evaluate the targeting effect of cyclic arginine-glycine-aspartic peptide (cRGD)-modified monomethoxy (polyethylene glycol)-poly (D, L-lactide-co-glycolide)-poly (L-lysine) nanoparticles (mPEG-PLGA-PLL-cRGD NPs) for gastric cancer SGC-7901 cells. We prepared the 5-Fulorouracil (5Fu)-loaded mPEG-PLGA-PLL-cRGD (5Fu/mPEG-PLGA-PLL-cRGD) NPs that had an average particle size of 180 nm and a zeta potential 2.77 mV. The results of cytotoxicity demonstrated the mPEG-PLGA-PLL-cRGD NPs showed the ignorable cytotoxicity and the 5Fu/mPEG-PLGA-PLL-cRGD NPs could significantly enhance the cytotoxicity of 5Fu. In vitro drug release experiments showed that the release of drug was effectively prolonged and sustained. The results of confocal laser scanning microscope (CLSM) and flow cytometer analysis demonstrated that the fluorescence intensity of the SGC-7901 gastric cancer cells treated with Rb/mPEG-PLGA-PLL-cRGD NPs was significantly higher than that treated with Rb, this suggested that Rb/mPEG-PLGA-PLL-cRGD NPs could effectively be internalized by SGC-7901 gastric cancer cells. In summary, the above experimental results illustrate that mPEG-PLGA-PLL-cRGD NPs have great potential to be used as an effective delivery carriers.

  4. Targeting retroviral and lentiviral vectors.

    PubMed

    Sandrin, V; Russell, S J; Cosset, F L

    2003-01-01

    Retroviral vectors capable of efficient in vivo gene delivery to specific target cell types or to specific locations of disease pathology would greatly facilitate many gene therapy applications. The surface glycoproteins of membrane-enveloped viruses stand among the choice candidates to control the target cell receptor recognition and host range of retroviral vectors onto which they are incorporated. This can be achieved in many ways, such as the exchange of glycoprotein by pseudotyping, their biochemical modifications, their conjugation with virus-cell bridging agents or their structural modifications. Understanding the fundamental properties of the viral glycoproteins and the molecular mechanism of virus entry into cells has been instrumental in the functional alteration of their tropism. Here we briefly review the current state of our understanding of the structure and function of viral envelope glycoproteins and we discuss the emerging targeting strategies based on retroviral and lentiviral vector systems.

  5. Targeting Adenoviral Vectors by Using the Extracellular Domain of the Coxsackie-Adenovirus Receptor: Improved Potency via Trimerization

    PubMed Central

    Kim, Jin; Smith,*, Theodore; Idamakanti, Neeraja; Mulgrew, Kathy; Kaloss, Michele; Kylefjord, Helen; Ryan, Patricia C.; Kaleko, Michael; Stevenson, Susan C.

    2002-01-01

    Adenovirus binds to mammalian cells via interaction of fiber with the coxsackie-adenovirus receptor (CAR). Redirecting adenoviral vectors to enter target cells via new receptors has the advantage of increasing the efficiency of gene delivery and reducing nonspecific transduction of untargeted tissues. In an attempt to reach this goal, we have produced bifunctional molecules with soluble CAR (sCAR), which is the extracellular domain of CAR fused to peptide-targeting ligands. Two peptide-targeting ligands have been evaluated: a cyclic RGD peptide (cRGD) and the receptor-binding domain of apolipoprotein E (ApoE). Human diploid fibroblasts (HDF) are poorly transduced by adenovirus due to a lack of CAR on the surface. Addition of the sCAR-cRGD or sCAR-ApoE targeting protein to adenovirus redirected binding to the appropriate receptor on HDF. However, a large excess of the monomeric protein was needed for maximal transduction, indicating a suboptimal interaction. To improve interaction of sCAR with the fiber knob, an isoleucine GCN4 trimerization domain was introduced, and trimerization was verified by cross-linking analysis. Trimerized sCAR proteins were significantly better at interacting with fiber and inhibiting binding to HeLa cells. Trimeric sCAR proteins containing cRGD and ApoE were more efficient at transducing HDF in vitro than the monomeric proteins. In addition, the trimerized sCAR protein without targeting ligands efficiently blocked liver gene transfer in normal C57BL/6 mice. However, addition of either ligand failed to retarget the liver in vivo. One explanation may be the large complex size, which serves to decrease the bioavailability of the trimeric sCAR-adenovirus complexes. In summary, we have demonstrated that trimerization of sCAR proteins can significantly improve the potency of this targeting approach in altering vector tropism in vitro and allow the efficient blocking of liver gene transfer in vivo. PMID:11799184

  6. cRGD grafted liposomes containing inorganic nano-precipitate complexed siRNA for intracellular delivery in cancer cells.

    PubMed

    Khatri, Nirav; Baradia, Dipesh; Vhora, Imran; Rathi, Mohan; Misra, Ambikanandan

    2014-05-28

    Development of effective vector for intracellular delivery of siRNA has always been a challenge due to its hydrophilicity, net negative surface charge and sensitivity against nucleases in biological milieu. The present investigation was aimed to develop a novel non-viral liposomal carrier for siRNA delivery. Nano-precipitate of calcium phosphate was entrapped in liposomes composed of a neutral lipid (DPPC), a fusogenic lipid (DOPE), a PEGylated lipid (DSPE-mPEG2000) and cholesterol. siRNA was made permeable through liposomal bilayer and complexed to calcium phosphate precipitates inside the liposomes. siRNA entrapped liposomes were further grafted with cRGD to achieve targeting potential against cancer cells. More than 80% of siRNA was entrapped inside the liposomes having average particle size below 150nm. Cryo-transmission electron microscopy revealed the intra-liposomal calcium phosphate precipitation and unilamellar morphology of prepared liposomes. The viability of A549 lung cancer cells was significantly higher after treatment with siRNA entrapped liposomes as compared to Lipofectamine2000 complexed siRNA. Fluorescent intensity in lung carcinoma cells was significantly higher after exposure to fluorescent siRNA entrapped liposomes than with Lipofectamine2000, which were confirmed by both confocal microscopy and flow cytometry. Live imaging by confocal microscopy ascertained the targeting efficacy of cRGD grafted liposomes compared to naked siRNA and non-grafted liposomes. Developed liposomal formulation showed effective protection of siRNA against serum nucleases along with less haemolytic potential and excellent stability against electrolyte induced flocculation. At 5nM concentration gene expression of target protein was reduced up to 24.1±3.4% while Lipofectamine2000 reduced expression level up to 26.35±1.55%. In vivo toxicity in mice suggested admirable safety profile for developed lipid based delivery vector. These results advocate that prepared

  7. Bacteriophage gene targeting vectors generated by transplacement.

    PubMed

    Aoyama, C; Woltjen, K; Mansergh, F C; Ishidate, K; Rancourt, D E

    2002-10-01

    A rate-determining step in gene targeting is the generation of the targeting vector. We have developed bacteriophage gene targeting vectorology, which shortens the timeline of targeting vector construction. Using retro-recombination screening, we can rapidly isolate targeting vectors from an embryonic stem cell genomic library via integrative and excisive recombination. We have demonstrated that recombination can be used to introduce specific point mutations or unique restriction sites into gene targeting vectors via transplacement. Using the choline/ethanolamine kinase alpha and beta genes as models, we demonstrate that transplacement can also be used to introduce specifically a neo resistance cassette into a gene targeting phage. In our experience, the lambdaTK gene targeting system offers considerable flexibility and efficiency in TV construction, which makes generating multiple vectors in one week's time possible.

  8. Engineering targeted viral vectors for gene therapy.

    PubMed

    Waehler, Reinhard; Russell, Stephen J; Curiel, David T

    2007-08-01

    To achieve therapeutic success, transfer vehicles for gene therapy must be capable of transducing target cells while avoiding impact on non-target cells. Despite the high transduction efficiency of viral vectors, their tropism frequently does not match the therapeutic need. In the past, this lack of appropriate targeting allowed only partial exploitation of the great potential of gene therapy. Substantial progress in modifying viral vectors using diverse techniques now allows targeting to many cell types in vitro. Although important challenges remain for in vivo applications, the first clinical trials with targeted vectors have already begun to take place.

  9. Vector quantization and learning vector quantization for radar target classification

    NASA Astrophysics Data System (ADS)

    Stewart, Clayton V.; Lu, Yi-Chuan; Larson, Victor J.

    1993-10-01

    Radar target classification performance is greatly dependent on how the classifier represents the strongly angle dependent radar target signatures. This paper compares the performance of classifiers that represent radar target signatures using vector quantization (VQ) and learning vector quantization (LVQ). The classifier performance is evaluated with a set of high resolution millimeter-wave radar data from four ground vehicles (Camaro, van, pickup, and bulldozer). LVQ explicitly includes classification performance in its data representation criterion, whereas VQ only makes use of a distortion measure such as mean square distance. The classifier that uses LVQ to represent the radar data has a much higher probability of correct classification than VQ.

  10. Sindbis viral vectors target hematopoietic malignant cells.

    PubMed

    Suzme, R; Tseng, J-C; Levin, B; Ibrahim, S; Meruelo, D; Pellicer, A

    2012-11-01

    Sindbis viral vectors target and inhibit the growth of various solid tumors in mouse models. However, their efficacy against blood cancer has not been well established. Here, we show that Sindbis vectors infect and efficiently trigger apoptosis in mouse BW5147 malignant hematopoietic T-cells, but only at low levels in human lymphoma and leukemia cells (Jurkat, Karpas, CEM, DHL and JB). The Mr 37/67 kD laminin receptor (LAMR) has been suggested to be the receptor for Sindbis virus. However, JB cells, which are infected by Sindbis at low efficiency, express high levels of LAMR, revealing that additional factors are involved in Sindbis tropism. To test the infectivity and therapeutic efficacy of Sindbis vectors against malignant hematopoietic cells in vivo, we injected BW5147 cells intraperitoneally into (C3HXAKR) F1 hybrid mice. We found that Sindbis vectors targeted the tumors and significantly prolonged survival of tumor-bearing mice. We also tested the Sindbis vectors in a transgenic CD4-Rgr model, which spontaneously develop thymic lymphomas. However, infectivity in this model was less efficient. Taken together, these results demonstrate that Sindbis vectors have the potential to target and kill hematopoietic malignancies in mice, but further research is needed to evaluate the mechanism underlining the susceptibility of human lymphoid malignancies to Sindbis therapy.

  11. Targeting lentiviral vectors for cancer immunotherapy

    PubMed Central

    Arce, Frederick; Breckpot, Karine; Collins, Mary; Escors, David

    2012-01-01

    Delivery of tumour-associated antigens (TAA) in a way that induces effective, specific immunity is a challenge in anti-cancer vaccine design. Circumventing tumour-induced tolerogenic mechanisms in vivo is also critical for effective immunotherapy. Effective immune responses are induced by professional antigen presenting cells, in particular dendritic cells (DC). This requires presentation of the antigen to both CD4+ and CD8+ T cells in the context of strong co-stimulatory signals. Lentiviral vectors have been tested as vehicles, for both ex vivo and in vivo delivery of TAA and/or activation signals to DC, and have been demonstrated to induce potent T cell mediated immune responses that can control tumour growth. This review will focus on the use of lentiviral vectors for in vivo gene delivery to DC, introducing strategies to target DC, either targeting cell entry or gene expression to improve safety of the lentiviral vaccine or targeting dendritic cell activation pathways to enhance performance of the lentiviral vaccine. In conclusion, this review highlights the potential of lentiviral vectors as a generally applicable ‘off-the-shelf’ anti-cancer immunotherapeutic. PMID:22983382

  12. Classification SAR targets with support vector machine

    NASA Astrophysics Data System (ADS)

    Cao, Lanying

    2007-02-01

    With the development of Synthetic Aperture Radar (SAR) technology, automatic target recognition (ATR) is becoming increasingly important. In this paper, we proposed a 3-class target classification system in SAR images. The system is based on invariant wavelet moments and support vector machine (SVM) algorithm. It is a two-stage approach. The first stage is to extract and select a small set of wavelet invariant moment features to indicate target images. The wavelet invariant moments take both advantages of the wavelet inherent property of multi-resolution analysis and moment invariants quality of invariant to translation, scaling changes and rotation. The second stage is classification of targets with SVM algorithm. SVM is based on the principle of structural risk minimization (SRM), which has been shown better than the principle of empirical risk minimization (ERM) which is used by many conventional networks. To test the performance and efficiency of the proposed method, we performed experiments on invariant wavelet moments, different kernel functions, 2-class identification, and 3-class identification. Test results show that wavelet invariant moments indicate the target effectively; linear kernel function achieves better results than other kernel functions, and SVM classification approach performs better than conventional nearest distance approach.

  13. Gold Nanorods Conjugated with Doxorubicin and cRGD for Combined Anticancer Drug Delivery and PET Imaging

    PubMed Central

    Xiao, Yuling; Hong, Hao; Matson, Vyara Z.; Javadi, Alireza; Xu, Wenjin; Yang, Yunan; Zhang, Yin; Engle, Jonathan W.; Nickles, Robert J.; Cai, Weibo; Steeber, Douglas A.; Gong, Shaoqin

    2012-01-01

    A multifunctional gold nanorod (GNR)-based nanoplatform for targeted anticancer drug delivery and positron emission tomography (PET) imaging of tumors was developed and characterized. An anti-cancer drug (i.e., doxorubicin (DOX)) was covalently conjugated onto PEGylated (PEG: polyethylene glycol) GNR nanocarriers via a hydrazone bond to achieve pH-sensitive controlled drug release. Tumor-targeting ligands (i.e., the cyclo(Arg-Gly-Asp-D-Phe-Cys) peptides, cRGD) and 64Cu-chelators (i.e., 1,4,7-triazacyclononane-N, N', N''-triacetic acid (NOTA)) were conjugated onto the distal ends of the PEG arms to achieve active tumor-targeting and PET imaging, respectively. Based on flow cytometry analysis, cRGD-conjugated nanocarriers (i.e., GNR-DOX-cRGD) exhibited a higher cellular uptake and cytotoxicity than non-targeted ones (i.e., GNR-DOX) in vitro. However, GNR-DOX-cRGD and GNR-DOX nanocarriers had similar in vivo biodistribution according to in vivo PET imaging and biodistribution studies. Due to the unique optical properties of GNRs, this multifunctional GNR-based nanoplatform can potentially be optimized for combined cancer therapies (chemotherapy and photothermal therapy) and multimodality imaging (PET, optical, X-ray computed tomography (CT), etc.). PMID:22916075

  14. Transductional targeting of adenovirus vectors for gene therapy

    PubMed Central

    Glasgow, JN; Everts, M; Curiel, DT

    2007-01-01

    Cancer gene therapy approaches will derive considerable benefit from adenovirus (Ad) vectors capable of self-directed localization to neoplastic disease or immunomodulatory targets in vivo. The ablation of native Ad tropism coupled with active targeting modalities has demonstrated that innate gene delivery efficiency may be retained while circumventing Ad dependence on its primary cellular receptor, the coxsackie and Ad receptor. Herein, we describe advances in Ad targeting that are predicated on a fundamental understanding of vector/cell interplay. Further, we propose strategies by which existing paradigms, such as nanotechnology, may be combined with Ad vectors to form advanced delivery vehicles with multiple functions. PMID:16439993

  15. Bacteriophage-Derived Vectors for Targeted Cancer Gene Therapy

    PubMed Central

    Pranjol, Md Zahidul Islam; Hajitou, Amin

    2015-01-01

    Cancer gene therapy expanded and reached its pinnacle in research in the last decade. Both viral and non-viral vectors have entered clinical trials, and significant successes have been achieved. However, a systemic administration of a vector, illustrating safe, efficient, and targeted gene delivery to solid tumors has proven to be a major challenge. In this review, we summarize the current progress and challenges in the targeted gene therapy of cancer. Moreover, we highlight the recent developments of bacteriophage-derived vectors and their contributions in targeting cancer with therapeutic genes following systemic administration. PMID:25606974

  16. A novel adenoviral vector labeled with superparamagnetic iron oxide nanoparticles for real-time tracking of viral delivery.

    PubMed

    Yun, Jonathan; Sonabend, Adam M; Ulasov, Ilya V; Kim, Dong-Hyun; Rozhkova, Elena A; Novosad, Valentyn; Dashnaw, Stephen; Brown, Truman; Canoll, Peter; Bruce, Jeffrey N; Lesniak, Maciej S

    2012-06-01

    In vivo tracking of gene therapy vectors challenges the investigation and improvement of biodistribution of these agents in the brain, a key feature for their targeting of infiltrative malignant gliomas. The glioma-targeting Ad5/3-cRGD gene therapy vector was covalently bound to super-paramagnetic iron oxide (Fe(3)O(4)) nanoparticles (SPION) to monitor its distribution by MRI. Transduction of labeled and unlabeled vectors was assessed on the U87 glioma cell line and normal human astrocytes (NHA), and was higher in U87 compared to NHA, but was similar between labeled and unlabeled virus. An in vivo study was performed by intracranial subcortical injection of labeled-Ad5/3-cRGD particles into a pig brain. The labeled vector appeared in vivo as a T2-weighted hyperintensity and a T2-gradient echo signal at the injection site, persisting up to 72 hours post-injection. We describe a glioma-targeting vector that is labeled with SPION, thereby allowing for MRI detection with no change in transduction capability. Copyright © 2012 Elsevier Ltd. All rights reserved.

  17. Radar target identification using probabilistic classification vector machines

    NASA Astrophysics Data System (ADS)

    Jouny, I.

    2016-05-01

    Radar target identification using probabilistic vector machines is investigated and tested using real radar data collected in a compact range for commercial aircraft models. Unlike relevance vector machines (RVM) that utilize zero-mean Gaussian prior for every weight for both negative and positive classes and are thus vulnerable to questionable (deceptive) vectors, probabilistic vector machines [2], alternatively, use nonnegative priors for the positive class and vice versa. This paper compares the performance of these machines with other target identification tools, and highlights scenarios where classification via a probabilistic vector machine is more plausible. The problem addressed in this paper is a M-ary target classification problem and is implemented as a set of pairwise comparisons between all competing hypotheses.

  18. Construction of gene-targeting vectors by recombineering.

    PubMed

    Lee, Song-Choon; Wang, Wei; Liu, Pentao

    2009-01-01

    Recombineering is a technology that utilizes the efficient homologous recombination functions encoded by gamma phage to manipulate DNA in Escherichia coli. Construction of knockout vectors has been greatly facilitated by recombineering as it allows one to choose any genomic region to manipulate. We describe here an efficient recombineering-based protocol for making mouse conditional knockout targeting vectors.

  19. Advances and Future Challenges in Adenoviral Vector Pharmacology and Targeting

    PubMed Central

    Khare, Reeti; Chen, Christopher Y; Weaver, Eric A; Barry, Michael A

    2011-01-01

    Adenovirus is a robust vector for therapeutic applications, but its use is limited by our understanding of its complex in vivo pharmacology. In this review we describe the necessity of identifying its natural, widespread, and multifaceted interactions with the host since this information will be crucial for efficiently redirecting virus into target cells. In the rational design of vectors, the notion of overcoming a sequence of viral “sinks” must be combined with re-targeting to target populations with capsid as well as shielding the vectors from pre-existing or toxic immune responses. It must also be noted that most known adenoviral pharmacology is deduced from the most commonly used serotypes, Ad5 and Ad2. However, these serotypes may not represent all adenoviruses, and may not even represent the most useful vectors for all purposes. Chimeras between Ad serotypes may become useful in engineering vectors that can selectively evade substantial viral traps, such as Kupffer cells, while retaining the robust qualities of Ad5. Similarly, vectorizing other Ad serotypes may become useful in avoiding immunity against Ad5 altogether. Taken together, this research on basic adenovirus biology will be necessary in developing vectors that interact more strategically with the host for the most optimal therapeutic effect. PMID:21453281

  20. Increasing the potential for malaria elimination by targeting zoophilic vectors

    PubMed Central

    Waite, Jessica L.; Swain, Sunita; Lynch, Penelope A.; Sharma, S. K.; Haque, Mohammed Asrarul; Montgomery, Jacqui; Thomas, Matthew B.

    2017-01-01

    Countries in the Asia Pacific region aim to eliminate malaria by 2030. A cornerstone of malaria elimination is the effective management of Anopheles mosquito vectors. Current control tools such as insecticide treated nets or indoor residual sprays target mosquitoes in human dwellings. We find in a high transmission region in India, malaria vector populations show a high propensity to feed on livestock (cattle) and rest in outdoor structures such as cattle shelters. We also find evidence for a shift in vector species complex towards increased zoophilic behavior in recent years. Using a malaria transmission model we demonstrate that in such regions dominated by zoophilic vectors, existing vector control tactics will be insufficient to achieve elimination, even if maximized. However, by increasing mortality in the zoophilic cycle, the elimination threshold can be reached. Current national vector control policy in India restricts use of residual insecticide sprays to domestic dwellings. Our study suggests substantial benefits of extending the approach to treatment of cattle sheds, or deploying other tactics that target zoophilic behavior. Optimizing use of existing tools will be essential to achieving the ambitious 2030 elimination target. PMID:28091570

  1. Optimizing targeted gene delivery: chemical modification of viral vectors and synthesis of artificial virus vector systems.

    PubMed

    Boeckle, Sabine; Wagner, Ernst

    2006-01-01

    In comparison to classical medicines, gene therapy has the potential to mediate the highest possible level of therapeutic specificity. Every normal or diseased cell can switch on or off a gene expression cassette in a tissue-, disease-, and time-dependent fashion, by use of specific transcription factors that are active only in a given unique situation. In practice, we face the problem in realizing the concept: the delivery of nucleic acids into target cells is very ineffective and presents a formidable challenge. Key issues for future developments include improved targeting, enhanced intracellular uptake, and reduced toxicity of gene vectors. The currently used classes of vectors have complementary characteristics, such as high intracellular efficiency of viral vectors on the one hand and low immunogenicity and greater flexibility of nonviral vectors on the other hand. The merge of viral and nonviral vector technologies is highlighted as an encouraging strategy for the future; concepts include chemically modified viral vectors ("chemo-viruses") and synthesis of virus-like systems ("synthetic viruses"). Examples for the development of vectors toward artificial synthetic viruses are presented.

  2. cRGD peptide installation on cisplatin-loaded nanomedicines enhances efficacy against locally advanced head and neck squamous cell carcinoma bearing cancer stem-like cells.

    PubMed

    Miyano, Kazuki; Cabral, Horacio; Miura, Yutaka; Matsumoto, Yu; Mochida, Yuki; Kinoh, Hiroaki; Iwata, Caname; Nagano, Osamu; Saya, Hideyuki; Nishiyama, Nobuhiro; Kataoka, Kazunori; Yamasoba, Tatsuya

    2017-09-10

    Recalcitrant head and neck squamous cell carcinoma (HNSCC) usually relapses after therapy due to the enrichment of drug resistant cancer stem-like cells (CSCs). Nanomedicines have shown potential for eradicating both cancer cells and CSCs by effective intratumoral navigation for reaching particular cell populations and controlling drug delivery. The installation of ligands on nanomedicines is an attractive approach for improving the delivery to CSCs within tumors, though the development of CSC-selective ligand-receptor systems has been challenging. Herein, we found that the CSC subpopulation in HNSCC cells overexpresses αvβ5 integrins, which is preferentially expressed in tumor neovasculature and cancer cells, and can be effectively targeted by using cyclic Arg-Gly-Asp (cRGD) peptide. Thus, in this study, we propose installing cRGD peptide on micellar nanomedicines incorporating cisplatin for improving their activity against CSCs and enhancing survival. Both cisplatin-loaded micelles (CDDP/m) and cRGD-installed CDDP/m (cRGD-CDDP/m) were effective against HNSCC SAS-L1-Luc cells in vitro, though cRGD-installed CDDP/m was more potent than CDDP/m against the CSC fraction. In vivo, the cRGD-CDDP/m also showed significant antitumor activity against HNSCC orthotopic tumors, i.e. SAS-L1 and HSC-2. Moreover, cRGD-CDDP/m rapidly accumulated into the lymph node metastasis of SAS-L1 tumors, effectively inhibiting their growth, and prolonging mice survival. These findings indicate cRGD-installed nanomedicines as an advantageous strategy for targeting CSCs in HNSCC, and particularly, cRGD-CDDP/m as a significant therapeutic strategy against regionally advanced HNSCC. Copyright © 2017. Published by Elsevier B.V.

  3. A target recognition algorithm based on a support vector machine

    NASA Astrophysics Data System (ADS)

    Ding, Yan; Jin, Weiqi; Yu, Yuhong; Wang, Han

    2008-12-01

    In order to meet the accuracy requirement of a target recognition system, a target recognition algorithm based on support vector machine is proposed in this paper. In the algorithm, firstly, a fast image multi-threshold segmentation method is accomplished by using a novel searching path of particle swarm optimization to separate the target from the background. Then some characteristics of target samples such as moment feature, affine invariant feature and texture feature based on co-occurrence matrix are extracted. Thus, the parameter optimizing selection is achieved according to the corresponding rule. After comparing with other kernel functions, the radial basis function kernel is selected to build a target classifier for one particular typical target. Meanwhile, a BP neural network based target recognition system is implemented to facilitate comparison. Finally, the target recognition method presented in this paper is applied to the airplane recognition. The experimental results show that the algorithm given in this paper can effectively detect and recognize the image target automatically. It can be applied to both single target and multi-objective recognition. Moreover, real-time target recognition can be achieved for single target.

  4. Peptide targeting of adenoviral vectors to augment tumor gene transfer.

    PubMed

    Ballard, E N; Trinh, V T; Hogg, R T; Gerard, R D

    2012-07-01

    Adenovirus serotype 5 remains one of the most promising vectors for delivering genetic material to cancer cells for imaging or therapy, but optimization of these agents to selectively promote tumor cell infection is needed to further their clinical development. Peptide sequences that bind to specific cell surface receptors have been inserted into adenoviral capsid proteins to improve tumor targeting, often in the background of mutations designed to ablate normal ligand:receptor interactions and thereby reduce off target effects and toxicities in non-target tissues. Different tumor types also express highly variable complements of cell surface receptors, so a customized targeting strategy using a particular peptide in the context of specific adenoviral mutations may be needed to achieve optimal efficacy. To further investigate peptide targeting strategies in adenoviral vectors, we used a set of peptide motifs originally isolated using phage display technology that evince tumor specificity in vivo. To demonstrate their abilities as targeting motifs, we genetically incorporated these peptides into a surface loop of the fiber capsid protein to construct targeted adenovirus vectors. We then systematically evaluated the ability of these peptide targeted vectors to infect several tumor cell types, both in vitro and in vivo, in a variety of mutational backgrounds designed to reduce CAR and/or HSG-mediated binding. Results from this study support previous observations that peptide insertions in the HI loop of the fiber knob domain are generally ineffective when used in combination with HSG detargeting mutations. The evidence also suggests that this strategy can attenuate other fiber knob interactions, such as CAR-mediated binding, and reduce overall viral infectivity. The insertion of peptides into fiber proved more effective for targeting tumor cell types expressing low levels of CAR receptor, as this strategy can partially compensate for the very low infectivity of wild

  5. Viral vectors for targeting the canine retina: a review.

    PubMed

    Petersen-Jones, Simon M

    2012-09-01

    Clinical trials are currently underway using gene therapy to treat retinal disease such as Leber congenital amaurosis (LCA). Viral vectors that have been utilized to target retinal cells include adenoviruses, lentiviruses, and recombinant adeno-associated viruses (rAAV). Of the three classes, rAAV vectors show the greatest promise for retinal gene therapy. Recent developments in virus technology such as the development of hybrid and capsid mutant rAAV vectors mean that specific retinal cells can be targeted and faster stronger transgene expression is now possible compared to that achieved with the first generation of vectors. Gene therapy trials in dogs have been very important in the development of therapy for RPE65 LCA which is currently in phase I/II clinical trials in humans. Recent successes in using gene therapy to treat canine achromatopsia, X-linked progressive retinal atrophy (PRA) and the more severe rapid degenerations such as rod-cone dysplasia type 3 may lead also to the translation to human clinical trials. Dogs have played and continue to play an important role as animal models for proof-of-concept studies of retinal gene therapy. As modifications and improvements in gene therapy protocols are made from experience gathered from human clinical trials perhaps gene therapy for the treatment of canine clinical patients will become available to veterinary ophthalmologists.

  6. Automatic Target Recognition Using Wavelet-Based Vector Quantization

    DTIC Science & Technology

    1997-12-01

    uses a set of dedicated vector quantizers (VQs) in the wavelet domain. The background pixels in each input image are properly clipped out by a set of...a target chip . . . . . . 8 5 Background clipping of several input images . . . . . . . . . . 8 6 Wavelet decomposition of a truck into four subbands...dedicated VQ for each subband within each aspect window. In the first stage, an aspect window is a background- clipping rectangle whose size is determined

  7. Targeting male mosquito swarms to control malaria vector density.

    PubMed

    Sawadogo, Simon Peguedwinde; Niang, Abdoulaye; Bilgo, Etienne; Millogo, Azize; Maïga, Hamidou; Dabire, Roch K; Tripet, Frederic; Diabaté, Abdoulaye

    2017-01-01

    Malaria control programs are being jeopardized by the spread of insecticide resistance in mosquito vector populations. It has been estimated that the spread of resistance could lead to an additional 120000 deaths per year, and interfere with the prospects for sustained control or the feasibility of achieving malaria elimination. Another complication for the development of resistance management strategies is that, in addition to insecticide resistance, mosquito behavior evolves in a manner that diminishes the impact of LLINs and IRS. Mosquitoes may circumvent LLIN and IRS control through preferential feeding and resting outside human houses and/or being active earlier in the evening before people go to sleep. Recent developments in our understanding of mosquito swarming suggest that new tools targeting mosquito swarms can be designed to cut down the high reproductive rate of malaria vectors. Targeting swarms of major malaria vectors may provide an effective control method to counteract behavioral resistance developed by mosquitoes. Here, we evaluated the impact of systematic spraying of swarms of Anopheles gambiae s.l. using a mixed carbamate and pyrethroid aerosol. The impact of this intervention on vector density, female insemination rates and the age structure of males was measured. We showed that the resulting mass killing of swarming males and some mate-seeking females resulted in a dramatic 80% decrease in population size compared to a control population. A significant decrease in female insemination rate and a significant shift in the age structure of the male population towards younger males incapable of mating were observed. This paradigm-shift study therefore demonstrates that targeting primarily males rather than females, can have a drastic impact on mosquito population.

  8. Targeting male mosquito swarms to control malaria vector density

    PubMed Central

    Sawadogo, Simon Peguedwinde; Niang, Abdoulaye; Bilgo, Etienne; Millogo, Azize; Maïga, Hamidou; Dabire, Roch K.; Tripet, Frederic; Diabaté, Abdoulaye

    2017-01-01

    Malaria control programs are being jeopardized by the spread of insecticide resistance in mosquito vector populations. It has been estimated that the spread of resistance could lead to an additional 120000 deaths per year, and interfere with the prospects for sustained control or the feasibility of achieving malaria elimination. Another complication for the development of resistance management strategies is that, in addition to insecticide resistance, mosquito behavior evolves in a manner that diminishes the impact of LLINs and IRS. Mosquitoes may circumvent LLIN and IRS control through preferential feeding and resting outside human houses and/or being active earlier in the evening before people go to sleep. Recent developments in our understanding of mosquito swarming suggest that new tools targeting mosquito swarms can be designed to cut down the high reproductive rate of malaria vectors. Targeting swarms of major malaria vectors may provide an effective control method to counteract behavioral resistance developed by mosquitoes. Here, we evaluated the impact of systematic spraying of swarms of Anopheles gambiae s.l. using a mixed carbamate and pyrethroid aerosol. The impact of this intervention on vector density, female insemination rates and the age structure of males was measured. We showed that the resulting mass killing of swarming males and some mate-seeking females resulted in a dramatic 80% decrease in population size compared to a control population. A significant decrease in female insemination rate and a significant shift in the age structure of the male population towards younger males incapable of mating were observed. This paradigm-shift study therefore demonstrates that targeting primarily males rather than females, can have a drastic impact on mosquito population. PMID:28278212

  9. Modification of pIX or hexon based on fiberless Ad vectors is not effective for targeted Ad vectors.

    PubMed

    Kurachi, Shinnosuke; Koizumi, Naoya; Tashiro, Katsuhisa; Sakurai, Haruna; Sakurai, Fuminori; Kawabata, Kenji; Nakagawa, Shinsaku; Mizuguchi, Hiroyuki

    2008-04-07

    Adenovirus (Ad) vector application in gene therapy is limited by its naïve tropism. We previously developed protein IX (pIX)-modified and hexon-modified Ad vectors in order to alter Ad vector tropism. However, these modified Ad vectors failed to infect cells with the foreign ligands displayed in the pIX or hexon. We hypothesized that steric hindrance by fiber proteins might have prevented the ligand-mediated transduction, as fibers are the outmost capsid proteins of Ad vectors. Therefore, we generated a series of fiberless Ad vectors and investigated their gene expression properties. Unexpectively, however, pIX- or hexon-modified fiberless Ad vector did not achieve any gene expression (the gene expression level by these vectors was similar to the background level). These results might be caused by the fact that the fiberless particles were weaker against physical burdens. To the best of our knowledge, this study is the first reported attempt to develop fiberless Ad vectors containing foreign ligands in the pIX or hexon region. The drawback of the lower stability of fiberless Ad vectors must be overcome to develop targeted Ad vectors based on such vectors. This study could provide basic information for the development of effective targeted Ad vectors.

  10. Bioengineered viral vectors for targeting and killing prostate cancer cells.

    PubMed

    Zhang, Kai-xin; Jia, William; Rennie, Paul S

    2010-01-01

    Enabling the transduction of therapeutic gene expression exclusively in diseased sites is the key to developing more effective treatments for advanced prostate cancer using viral-based therapy. While prostate cancers that express high levels of HER-2 are resistant to the killing effects of trastuzumab, they can be targeted for selective gene expression and destruction by lentiviruses with envelope proteins engineered to bind to this therapeutic antibody. More importantly, after intravenous injection, this trastuzumab-bound lentivirus is able to target castration-resistant prostate tumor xenografts, albeit with low efficiency. This proof of principle opens up multiple possibilities for the prevention and treatment of prostate cancer using a viral-based therapy. However, to be safe and more effective, the viral vectors must target prostate cancer cells more selectively and efficiently. A higher degree of specificity and efficiency of cancer cell targeting can be achieved by engineering viral vectors to bind to a specific cell surface marker and by controlling the expression of the therapeutic payload at transcriptional level, with a tissue-specific promoter, and at the translational level, with a regulatory sequences inserted into either the 5'UTR or 3'UTR regions of the therapeutic gene(s). The latter would be designed to ensure that translation of this mRNA occurs exclusively in malignant cells. Furthermore, in order to obtain a potent anti-tumor effect, viral vectors would be engineered to express pro-apoptotic genes, intra-cellar antibodies/nucleotide aptamers to block critical proteins, or siRNAs to knockdown essential cellular mRNAs. Alternatively, controlled expression of an essential viral gene would restore replication competence to the virus and enable selective oncolysis of tumor cells. Successful delivery of such bioengineered viruses may provide a more effective way to treat advanced prostate cancer.

  11. Library screening and receptor-directed targeting of gammaretroviral vectors.

    PubMed

    Mazari, Peter M; Roth, Monica J

    2013-01-01

    Gene- and cell-based therapies hold great potential for the advancement of the personalized medicine movement. Gene therapy vectors have made dramatic leaps forward since their inception. Retroviral-based vectors were the first to gain clinical attention and still offer the best hope for the long-term correction of many disorders. The fear of nonspecific transduction makes targeting a necessary feature for most clinical applications. However, this remains a difficult feature to optimize, with specificity often coming at the expense of efficiency. The aim of this article is to discuss the various methods employed to retarget retroviral entry. Our focus will lie on the modification of gammaretroviral envelope proteins with an in-depth discussion of the creation and screening of envelope libraries.

  12. Efficiency of insertion versus replacement vector targeting varies at different chromosomal loci.

    PubMed Central

    Hasty, P; Crist, M; Grompe, M; Bradley, A

    1994-01-01

    We have analyzed the targeting frequencies and recombination products generated with isogenic vectors at the fah and fgr loci in embryonic stem cells. A single vector which could be linearized at different sites to generate either a replacement or an insertion vector was constructed for each locus. A replacement event predominated when the vectors were linearized at the edge of the homologous sequences, while an insertion event predominated when the vectors were linearized within the homologous sequences. However, the ratio of the targeting frequencies exhibited by the different vector configurations differed for the two loci. When the fgr vector was linearized as an insertion vector, the ratio of targeted to random integrations was four- to eightfold greater than when the vector was linearized as a replacement vector. By contrast, the ratio of targeted to random integrations at the fah locus did not vary with the linearization site of the vector. The different relationships between the targeting frequency and the vector configuration at the fgr and fah loci may indicate a DNA sequence or chromatin structure preference for different targeting pathways. Images PMID:7969173

  13. Genetic modification of hematopoietic cells using retroviral and lentiviral vectors: safety considerations for vector design and delivery into target cells.

    PubMed

    Dropulic, Boro

    2005-07-01

    The recent development of leukemia in three patients following retroviral vector gene transfer in hematopoietic stem cells, resulting in the death of one patient, has raised safety concerns for the use of integrating gene transfer vectors for human gene therapy. This review discusses these serious adverse events from the perspective of whether restrictions on vector design and vector-modified target cells are warranted at this time. A case is made against presently establishing specific restrictions for vector design and transduced cells; rather, their safety should be ascertained by empiric evaluation in appropriate preclinical models on a case-by-case basis. Such preclinical data, coupled with proper informed patient consent and a risk-benefit ratio analysis, provide the best available prospective evaluation of gene transfer vectors prior to their translation into the clinic.

  14. Efficient construction of rAAV-based gene targeting vectors by Golden Gate cloning.

    PubMed

    Luo, Yonglun; Lin, Lin; Bolund, Lars; Sørensen, Charlotte Brandt

    2014-05-01

    The recombinant adeno-associated virus (rAAV) has proven to be an efficient and attractive tool for targeted genome engineering. Here we present a novel method employing the Golden Gate cloning strategy for fast and efficient construction of rAAV-based gene knockout or single-nucleotide knockin vectors. Two vectors, pGolden-Neo and pGolden-Hyg, were generated as common assembling modules to confer antibiotic resistance to the targeting vector. To validate the method, we then generated two rAAV-based targeting vectors: pAAV-pTP53-KO and pAAV-hTau(P301L)-KI. Furthermore, we generated a pGolden-AAV plasmid that allows one-step generation of an rAAV-based targeting vector. Our new methodology for rAAV targeting vector assembly is efficient, accurate, time-saving, and cost-effective.

  15. Adenovirus Vectors Targeting Distinct Cell Types in the Retina

    PubMed Central

    Sweigard, J. Harry; Cashman, Siobhan M.

    2010-01-01

    Purpose. Gene therapy for a number of retinal diseases necessitates efficient transduction of photoreceptor cells. Whereas adenovirus (Ad) serotype 5 (Ad5) does not transduce photoreceptors efficiently, previous studies have demonstrated improved photoreceptor transduction by Ad5 pseudotyped with Ad35 (Ad5/F35) or Ad37 (Ad5/F37) fiber or by the deletion of the RGD domain in the Ad5 penton base (Ad5ΔRGD). However, each of these constructs contained a different transgene cassette, preventing the evaluation of the relative performance of these vectors, an important consideration before the use of these vectors in the clinic. The aim of this study was to evaluate these vectors in the retina and to attempt photoreceptor-specific transgene expression. Methods. Three Ad5-based vectors containing the same expression cassette were generated and injected into the subretinal space of adult mice. Eyes were analyzed for green fluorescence protein expression in flat-mounts, cross-sections, quantitative RT-PCR, and a modified stereological technique. A 257-bp fragment derived from the mouse opsin promoter was analyzed in the context of photoreceptor-specific transgene expression. Results. Each virus tested efficiently transduced the retinal pigment epithelium. The authors found no evidence that Ad5/F35 or Ad5/F37 transduced photoreceptors. Instead, they found that Ad5/F37 transduced Müller cells. Robust photoreceptor transduction by Ad5ΔRGD was detected. Photoreceptor-specific transgene expression from the 257-bp mouse opsin promoter in the context of Ad5ΔRGD vectors was found. Conclusions. Adenovirus vectors may be designed with tropism to distinct cell populations. Robust photoreceptor-specific transgene expression can be achieved in the context of Ad5ΔRGD vectors. PMID:19892875

  16. Avidin-Based Targeting and Purification of a Protein IX-Modified, Metabolically Biotinylated Adenoviral Vector

    PubMed Central

    Campos, Samuel K.; Parrott, M. Brandon; Barry, Michael A.

    2014-01-01

    While genetic modification of adenoviral vectors can produce vectors with modified tropism, incorporation of targeting peptides/proteins into the structural context of the virion can also result in destruction of ligand targeting or virion integrity. To combat this problem, we have developed a versatile targeting system using metabolically biotinylated adenoviral vectors bearing biotinylated fiber proteins. These vectors have been demonstrated to be useful as a platform for avidin-based ligand screening and vector targeting by conjugating biotinylated ligands to the virus using high-affinity tetrameric avidin (Kd = 10−15 M). The biotinylated vector could also be purified by biotin-reversible binding on monomeric avidin (Kd = 10−7 M). In this report, a second metabolically biotinylated adenovirus vector, Ad-IX-BAP, has been engineered by fusing a biotin acceptor peptide (BAP) to the C-terminus of the adenovirus pIX protein. This biotinylated vector displays twice as many biotins and was markedly superior for single-step affinity purification on monomeric avidin resin. However, unlike the fiber-biotinylated vector, Ad-IX-BAP failed to retarget to cells with biotinylated antibodies including anti-CD71 against the transferrin receptor. In contrast, Ad-IX-BAP was retargeted if transferrin, the cognate ligand for CD71, was used as a ligand rather than the anti-CD71. This work demonstrates the utility of metabolic biotinylation as a molecular screening tool to assess the utility of different viral capsid proteins for ligand display and the biology and compatibility of different ligands and receptors for vector targeting applications. These results also demonstrate the utility of the pIX-biotinylated vector as a platform for gentle single-step affinity purification of adenoviral vectors. PMID:15194061

  17. Development of Lentiviral Vectors for Targeted Integration and Protein Delivery.

    PubMed

    Schenkwein, Diana; Ylä-Herttuala, Seppo

    2016-01-01

    The method in this chapter describes the design of human immunodeficiency virus type 1 (HIV-1) integrase (IN)-fusion proteins which we have developed to transport different proteins into the nuclei of lentiviral vector (LV)-transduced cells. The IN-fusion protein cDNA is incorporated into the LV packaging plasmid, which leads to its incorporation into vector particles as part of a large Gag-Pol polyprotein. This specific feature of protein packaging enables also the incorporation of cytotoxic and proapoptotic proteins, such as frequently cutting endonucleases and P53. The vectors can hence be used for various protein transduction needs. An outline of the necessary methods is also given to study the functionality of a chosen IN-fusion protein in a cell culture assay.

  18. Vector-transmitted disease vaccines: targeting salivary proteins in transmission (SPIT).

    PubMed

    McDowell, Mary Ann

    2015-08-01

    More than half the population of the world is at risk for morbidity and mortality from vector-transmitted diseases, and emerging vector-transmitted infections are threatening new populations. Rising insecticide resistance and lack of efficacious vaccines highlight the need for novel control measures. One such approach is targeting the vector-host interface by incorporating vector salivary proteins in anti-pathogen vaccines. Debate remains about whether vector saliva exposure exacerbates or protects against more severe clinical manifestations, induces immunity through natural exposure or extends to all vector species and associated pathogens. Nevertheless, exploiting this unique biology holds promise as a viable strategy for the development of vaccines against vector-transmitted diseases.

  19. Direct selection of targeted adenovirus vectors by random peptide display on the fiber knob.

    PubMed

    Miura, Y; Yoshida, K; Nishimoto, T; Hatanaka, K; Ohnami, S; Asaka, M; Douglas, J T; Curiel, D T; Yoshida, T; Aoki, K

    2007-10-01

    Targeting of gene transfer at the level of cell entry is one of the most attractive challenges in vector development. However, attempts to redirect adenovirus vectors to alternative receptors by engineering the capsid-coding region have shown limited success because proper targeting ligand-receptor systems on the cells of interest are generally unknown. Systematic approaches to generate adenovirus vectors targeting any given cell type need to be developed to achieve this goal. Here, we constructed an adenovirus library that was generated by a Cre-lox-mediated in vitro recombination between an adenoviral fiber-modified plasmid library and genomic DNA to display random peptides on a fiber knob. As proof of concept, we screened the adenovirus display library on a glioma cell line and observed selection of several particular peptide sequences. The targeted vector carrying the most frequently isolated peptide significantly enhanced gene transduction in the glioma cell line but not in many other cell lines. Because the insertion of a pre-selected peptide into a fiber knob often fails to generate an adenovirus vector, the selection of targeting peptides is highly useful in the context of the adenoviral capsid. This vector-screening system can facilitate the development of a targeted adenovirus vector for a variety of applications in medicine.

  20. Targeted systemic gene therapy and molecular imaging of cancer contribution of the vascular-targeted AAVP vector.

    PubMed

    Hajitou, Amin

    2010-01-01

    Gene therapy and molecular-genetic imaging have faced a major problem: the lack of an efficient systemic gene delivery vector. Unquestionably, eukaryotic viruses have been the vectors of choice for gene delivery to mammalian cells; however, they have had limited success in systemic gene therapy. This is mainly due to undesired uptake by the liver and reticuloendothelial system, broad tropism for mammalian cells causing toxicity, and their immunogenicity. On the other hand, prokaryotic viruses such as bacteriophage (phage) have no tropism for mammalian cells, but can be engineered to deliver genes to these cells. However, phage-based vectors have inherently been considered poor vectors for mammalian cells. We have reported a new generation of vascular-targeted systemic hybrid prokaryotic-eukaryotic vectors as chimeras between an adeno-associated virus (AAV) and targeted bacteriophage (termed AAV/phage; AAVP). In this hybrid vector, the targeted bacteriophage serves as a shuttle to deliver the AAV transgene cassette inserted in an intergenomic region of the phage DNA genome. As a proof of concept, we assessed the in vivo efficacy of vector in animal models of cancer by displaying on the phage capsid the cyclic Arg-Gly-Asp (RGD-4C) ligand that binds to alphav integrin receptors specifically expressed on the angiogenic blood vessels of tumors. The ligand-directed vector was able to specifically deliver imaging and therapeutic transgenes to tumors in mice, rats, and dogs while sparing the normal organs. This chapter reviews some gene transfer strategies and the potential of the vascular-targeted AAVP vector for enhancing the effectiveness of existing systemic gene delivery and genetic-imaging technologies.

  1. Enhanced Peptide of Prostate Cancer Using Targeted Adenoviral Vectors

    DTIC Science & Technology

    2005-06-01

    greater than that observed in tumors injected with control adenovirus (1.4 - 1..6% ID/g). Another adenovirus encoding for both SSTR2 and cytosine deaminase ...for treating prostate cancer xenografts which involves the use of an adenoviral vector encoding for both SSTR2 and the cytosine deaminase (CD) enzyme...SSTR2 and bacterial cytosine deaminase (CD) was performed in a manner similar to that previously described. The AdEasy system was used to generate the

  2. A series of conditional shuttle vectors for targeted genomic integration in budding yeast

    PubMed Central

    Chou, Chia-Ching; Patel, Michael T.; Gartenberg, Marc R.

    2015-01-01

    The capacity of Saccharomyces cerevisiae to repair exposed DNA ends by homologous recombination has long been used by experimentalists to assemble plasmids from DNA fragments in vivo. While this approach works well for engineering extrachromosomal vectors, it is not well suited to the generation, recovery and reuse of integrative vectors. Here, we describe the creation of a series of conditional centromeric shuttle vectors, termed pXR vectors, that can be used for both plasmid assembly in vivo and targeted genomic integration. The defining feature of pXR vectors is that the DNA segment bearing the centromere and origin of replication, termed CEN/ARS, is flanked by a pair of loxP sites. Passaging the vectors through bacteria that express Cre recombinase reduces the loxP-CEN/ARS-loxP module to a single loxP site, thereby eliminating the ability to replicate autonomously in yeast. Each vector also contains a selectable marker gene, as well as a fragment of the HO locus, which permits targeted integration at a neutral genomic site. The pXR vectors provide a convenient and robust method to assemble DNAs for targeted genomic modifications. PMID:25736914

  3. A series of conditional shuttle vectors for targeted genomic integration in budding yeast.

    PubMed

    Chou, Chia-Ching; Patel, Michael T; Gartenberg, Marc R

    2015-05-01

    The capacity of Saccharomyces cerevisiae to repair exposed DNA ends by homologous recombination has long been used by experimentalists to assemble plasmids from DNA fragments in vivo. While this approach works well for engineering extrachromosomal vectors, it is not well suited to the generation, recovery and reuse of integrative vectors. Here, we describe the creation of a series of conditional centromeric shuttle vectors, termed pXR vectors, that can be used for both plasmid assembly in vivo and targeted genomic integration. The defining feature of pXR vectors is that the DNA segment bearing the centromere and origin of replication, termed CEN/ARS, is flanked by a pair of loxP sites. Passaging the vectors through bacteria that express Cre recombinase reduces the loxP-CEN/ARS-loxP module to a single loxP site, thereby eliminating the ability to replicate autonomously in yeast. Each vector also contains a selectable marker gene, as well as a fragment of the HO locus, which permits targeted integration at a neutral genomic site. The pXR vectors provide a convenient and robust method to assemble DNAs for targeted genomic modifications.

  4. Advances in the Development of Gene-Targeting Vectors to Increase the Efficiency of Genetic Modification.

    PubMed

    Saito, Shinta; Adachi, Noritaka

    2016-01-01

    Gene targeting via homologous recombination, albeit highly inefficient in human cells, is considered a powerful tool for analyzing gene functions. Despite recent progress in the application of artificial nucleases for genome editing, safety issues remain a concern, particularly when genetic modification is used for therapeutic purposes. Therefore, the development of gene-targeting vectors is necessary for safe and sophisticated genetic modification. In this paper, we describe the effect of vector structure on random integration, which is a major obstacle in efficient gene targeting. In addition, we focus on the features of exon-trapping-type gene-targeting vectors, and discuss a novel strategy for negative selection to enhance gene targeting in human cells.

  5. Rabies virus envelope glycoprotein targets lentiviral vectors to the axonal retrograde pathway in motor neurons.

    PubMed

    Hislop, James N; Islam, Tarin A; Eleftheriadou, Ioanna; Carpentier, David C J; Trabalza, Antonio; Parkinson, Michael; Schiavo, Giampietro; Mazarakis, Nicholas D

    2014-06-06

    Rabies pseudotyped lentiviral vectors have great potential in gene therapy, not least because of their ability to transduce neurons following their distal axonal application. However, very little is known about the molecular processes that underlie their retrograde transport and cell transduction. Using multiple labeling techniques and confocal microscopy, we demonstrated that pseudotyping with rabies virus envelope glycoprotein (RV-G) enabled the axonal retrograde transport of two distinct subtypes of lentiviral vector in motor neuron cultures. Analysis of this process revealed that these vectors trafficked through Rab5-positive endosomes and accumulated within a non-acidic Rab7 compartment. RV-G pseudotyped vectors were co-transported with both the tetanus neurotoxin-binding fragment and the membrane proteins thought to mediate rabies virus endocytosis (neural cell adhesion molecule, nicotinic acetylcholine receptor, and p75 neurotrophin receptor), thus demonstrating that pseudotyping with RV-G targets lentiviral vectors for transport along the same pathway exploited by several toxins and viruses. Using motor neurons cultured in compartmentalized chambers, we demonstrated that axonal retrograde transport of these vectors was rapid and efficient; however, it was not able to transduce the targeted neurons efficiently, suggesting that impairment in processes occurring after arrival of the viral vector in the soma is responsible for the low transduction efficiency seen in vivo, which suggests a novel area for improvement of gene therapy vectors.

  6. Stabilization of Structured Populations via Vector Target-Oriented Control.

    PubMed

    Braverman, Elena; Franco, Daniel

    2017-08-01

    In contrast with unstructured models, structured discrete population models have been able to fit and predict chaotic experimental data. However, most of the chaos control techniques in the literature have been designed and analyzed in a one-dimensional setting. Here, by introducing target-oriented control for discrete dynamical systems, we prove the possibility to stabilize a chosen state for a wide range of structured population models. The results are illustrated with introducing a control in the celebrated LPA model describing a flour beetle dynamics. Moreover, we show that the new control allows to stabilize periodic solutions for higher-order difference equations, such as the delayed Ricker model, for which previous target-oriented methods were not designed.

  7. Target versus background characterization: second-generation wavelets and support vector machines

    NASA Astrophysics Data System (ADS)

    Gorsich, David J.; Karlsen, Robert E.; Gerhart, Grant R.; Genton, Marc G.

    1999-07-01

    The problem of determining the difference between a target and the background is a very difficult and ill-posed problem, yet it is a problem constantly faced by engineers working in target detection and machine vision. Terms like target, background, and clutter are not well defined and are often used differently in every context. Clutter can be defined as a stationary noise process, anything non-target, or anything that looks like a target but is not. Targets can be defined by deformable templates, models, or by specific feature vectors. Models, templates and features must be defined before classification begins. Both models and feature vectors somehow hold the defining characteristics of the target, for example the gun barrel of a tank. Most importantly, feature vectors and models reduce the dimensionality of the problem making numerical methods possible. This paper explores several fairly recent techniques that provide promising new approaches to these old problems. Wavelets are used to de-trend images to eliminate deterministic components, and a trained support vector machine is used to classify the remaining complicated or stochastic components of the image. Ripely's K-function is used to study the spatial location of the wavelet coefficients. The support vector machine avoids the choice of a model or feature vector, and the wavelets provide a way to determine the non-predictability of the local image components. The K-function of the wavelet coefficients serves as a new clutter metric. The technique is tested on the TNO image set through several random simulations.

  8. Cells as vehicles for cancer gene therapy: the missing link between targeted vectors and systemic delivery?

    PubMed

    Harrington, Kevin; Alvarez-Vallina, Luis; Crittenden, Marka; Gough, Michael; Chong, Heung; Diaz, Rosa Maria; Vassaux, Georges; Lemoine, Nicholas; Vile, Richard

    2002-07-20

    Systemic administration of currently manufactured viral stocks has not so far achieved sufficient circulating titers to allow therapeutic targeting of metastatic disease. This is due to low initial viral titers, immune inactivation, nonspecific adhesion, and loss of particles. One way to exploit the elegant molecular manipulations that have been made to increase vector targeting is to protect these vectors until they reach the local sites of tumor growth. Various cell types home preferentially to tumors and can be loaded with the constructs required to produce targeted vectors. Here we discuss the potential of using such cell carriers to chaperone precious vectors directly to the tumors. The vectors can incorporate mechanisms to achieve tumor site-inducible expression, along with tumor cell-specific expression of the therapeutic gene and/or replicating viral genomes that would be released at the tumor. In this way, the great advances that have so far been made with the engineering of vector tropisms might be genuinely exploited and converted into clinical benefit.

  9. Multiplexed Targeted Genome Engineering Using a Universal Nuclease-Assisted Vector Integration System.

    PubMed

    Brown, Alexander; Woods, Wendy S; Perez-Pinera, Pablo

    2016-07-15

    Engineered nucleases are capable of efficiently modifying complex genomes through introduction of targeted double-strand breaks. However, mammalian genome engineering remains limited by low efficiency of heterologous DNA integration at target sites, which is typically performed through homologous recombination, a complex, ineffective and costly process. In this study, we developed a multiplexable and universal nuclease-assisted vector integration system for rapid generation of gene knock outs using selection that does not require customized targeting vectors, thereby minimizing the cost and time frame needed for gene editing. Importantly, this system is capable of remodeling native mammalian genomes through integration of DNA, up to 50 kb, enabling rapid generation and screening of multigene knockouts from a single transfection. These results support that nuclease assisted vector integration is a robust tool for genome-scale gene editing that will facilitate diverse applications in synthetic biology and gene therapy.

  10. Specific Retrograde Transduction of Spinal Motor Neurons Using Lentiviral Vectors Targeted to Presynaptic NMJ Receptors

    PubMed Central

    Eleftheriadou, I; Trabalza, A; Ellison, SM; Gharun, K; Mazarakis, ND

    2014-01-01

    To understand how receptors are involved in neuronal trafficking and to be able to utilize them for specific targeting via the peripheral route would be of great benefit. Here, we describe the generation of novel lentiviral vectors with tropism to motor neurons that were made by coexpressing onto the lentiviral surface a fusogenic glycoprotein (mutated sindbis G) and an antibody against a cell-surface receptor (Thy1.1, p75NTR, or coxsackievirus and adenovirus receptor) on the presynaptic terminal of the neuromuscular junction. These vectors exhibit binding specificity and efficient transduction of receptor positive cell lines and primary motor neurons in vitro. Targeting of each of these receptors conferred to these vectors the capability of being transported retrogradely from the axonal tip, leading to transduction of motor neurons in vitro in compartmented microfluidic cultures. In vivo delivery of coxsackievirus and adenovirus receptor-targeted vectors in leg muscles of mice resulted in predicted patterns of motor neuron labeling in lumbar spinal cord. This opens up the clinical potential of these vectors for minimally invasive administration of central nervous system-targeted therapeutics in motor neuron diseases. PMID:24670531

  11. The research and application of visual saliency and adaptive support vector machine in target tracking field.

    PubMed

    Chen, Yuantao; Xu, Weihong; Kuang, Fangjun; Gao, Shangbing

    2013-01-01

    The efficient target tracking algorithm researches have become current research focus of intelligent robots. The main problems of target tracking process in mobile robot face environmental uncertainty. They are very difficult to estimate the target states, illumination change, target shape changes, complex backgrounds, and other factors and all affect the occlusion in tracking robustness. To further improve the target tracking's accuracy and reliability, we present a novel target tracking algorithm to use visual saliency and adaptive support vector machine (ASVM). Furthermore, the paper's algorithm has been based on the mixture saliency of image features. These features include color, brightness, and sport feature. The execution process used visual saliency features and those common characteristics have been expressed as the target's saliency. Numerous experiments demonstrate the effectiveness and timeliness of the proposed target tracking algorithm in video sequences where the target objects undergo large changes in pose, scale, and illumination.

  12. Targeting renal epithelial channels for the control of insect vectors

    PubMed Central

    Beyenbach, Klaus W; Yu, Yasong; Piermarini, Peter M; Denton, Jerod

    2015-01-01

    Three small molecules were identified in high throughput screens that 1) block renal inward rectifier potassium (Kir) channels of Aedes aegypti expressed in HEK cells and Xenopus oocytes, 2) inhibit the secretion of KCl but not NaCl in isolated Malpighian tubules, and after injection into the hemolymph, 3) inhibit KCl excretion in vivo, and 4) render mosquitoes flightless or dead within 24h. Some mosquitoes had swollen abdomens at death consistent with renal failure. VU625, the most potent and promising small molecule for development as mosquitocide, inhibits AeKir1-mediated currents with an IC50 less than 100 nM. It is highly selective for AeKir1 over mammalian Kir channels, and it affects only 3 of 68 mammalian membrane proteins. These results document 1) renal failure as a new mode-of-action for mosquitocide development, 2) renal Kir channels as molecular target for inducing renal failure, and 3) the promise of the discovery and development of new species-specific insecticides. PMID:26716074

  13. Adenovirus Vectors Target Several Cell Subtypes of Mammalian Inner Ear In Vivo

    PubMed Central

    Li, Wenyan; Shen, Jun

    2016-01-01

    Mammalian inner ear harbors diverse cell types that are essential for hearing and balance. Adenovirus is one of the major vectors to deliver genes into the inner ear for functional studies and hair cell regeneration. To identify adenovirus vectors that target specific cell subtypes in the inner ear, we studied three adenovirus vectors, carrying a reporter gene encoding green fluorescent protein (GFP) from two vendors or with a genome editing gene Cre recombinase (Cre), by injection into postnatal days 0 (P0) and 4 (P4) mouse cochlea through scala media by cochleostomy in vivo. We found three adenovirus vectors transduced mouse inner ear cells with different specificities and expression levels, depending on the type of adenoviral vectors and the age of mice. The most frequently targeted region was the cochlear sensory epithelium, including auditory hair cells and supporting cells. Adenovirus with GFP transduced utricular supporting cells as well. This study shows that adenovirus vectors are capable of efficiently and specifically transducing different cell types in the mammalian inner ear and provides useful tools to study inner ear gene function and to evaluate gene therapy to treat hearing loss and vestibular dysfunction. PMID:28116172

  14. Retargeting vesicular stomatitis virus glycoprotein pseudotyped lentiviral vectors with enhanced stability by in situ synthesized polymer shell.

    PubMed

    Liang, Min; Yan, Ming; Lu, Yunfeng; Chen, Irvin S Y

    2013-02-01

    The ability to introduce transgenes with precise specificity to the desired target cells or tissues is key to a more facile application of genetic therapy. Here, we describe a novel method using nanotechnology to generate lentiviral vectors with altered recognition of host cell receptor specificity. Briefly, the infectivity of the vesicular stomatitis virus glycoprotein (VSV-G) pseudotyped lentiviral vectors was shielded by a thin polymer shell synthesized in situ onto the viral envelope, and new binding ability was conferred to the shielded virus by introducing acrylamide-tailored cyclic arginine-glycine-aspartic acid (cRGD) peptide to the polymer shell. We termed the resulting virus "targeting nanovirus." The targeting nanovirus had similar titer with VSV-G pseudotypes and specifically transduced Hela cells with high transduction efficiency. In addition, the encapsulation of the VSV-G pseudotyped lentivirus by the polymer shell did not change the pathway that VSV-G pseudotypes enter and fuse with cells, as well as later events such as reverse transcription and gene expression. Furthermore, the targeting nanovirus possessed enhanced stability in the presence of human serum, indicating protection of the virus by the polymer shell from human serum complement inactivation. This novel use of nanotechnology demonstrates proof of concept for an approach that could be more generally applied for redirecting viral vectors for laboratory and clinical purposes.

  15. Retargeting Vesicular Stomatitis Virus Glycoprotein Pseudotyped Lentiviral Vectors with Enhanced Stability by In Situ Synthesized Polymer Shell

    PubMed Central

    Liang, Min; Yan, Ming; Lu, Yunfeng

    2013-01-01

    Abstract The ability to introduce transgenes with precise specificity to the desired target cells or tissues is key to a more facile application of genetic therapy. Here, we describe a novel method using nanotechnology to generate lentiviral vectors with altered recognition of host cell receptor specificity. Briefly, the infectivity of the vesicular stomatitis virus glycoprotein (VSV-G) pseudotyped lentiviral vectors was shielded by a thin polymer shell synthesized in situ onto the viral envelope, and new binding ability was conferred to the shielded virus by introducing acrylamide-tailored cyclic arginine-glycine-aspartic acid (cRGD) peptide to the polymer shell. We termed the resulting virus “targeting nanovirus.” The targeting nanovirus had similar titer with VSV-G pseudotypes and specifically transduced Hela cells with high transduction efficiency. In addition, the encapsulation of the VSV-G pseudotyped lentivirus by the polymer shell did not change the pathway that VSV-G pseudotypes enter and fuse with cells, as well as later events such as reverse transcription and gene expression. Furthermore, the targeting nanovirus possessed enhanced stability in the presence of human serum, indicating protection of the virus by the polymer shell from human serum complement inactivation. This novel use of nanotechnology demonstrates proof of concept for an approach that could be more generally applied for redirecting viral vectors for laboratory and clinical purposes. PMID:23327104

  16. Evaluation of Spatially Targeted Strategies to Control Non-Domiciliated Triatoma dimidiata Vector of Chagas Disease

    PubMed Central

    Barbu, Corentin; Dumonteil, Eric; Gourbière, Sébastien

    2011-01-01

    Background Chagas disease is a major neglected tropical disease with deep socio-economical effects throughout Central and South America. Vector control programs have consistently reduced domestic populations of triatomine vectors, but non-domiciliated vectors still have to be controlled efficiently. Designing control strategies targeting these vectors is challenging, as it requires a quantitative description of the spatio-temporal dynamics of village infestation, which can only be gained from combinations of extensive field studies and spatial population dynamic modelling. Methodology/Principal Findings A spatially explicit population dynamic model was combined with a two-year field study of T. dimidiata infestation dynamics in the village of Teya, Mexico. The parameterized model fitted and predicted accurately both intra-annual variation and the spatial gradient in vector abundance. Five different control strategies were then applied in concentric rings to mimic spatial design targeting the periphery of the village, where vectors were most abundant. Indoor insecticide spraying and insect screens reduced vector abundance by up to 80% (when applied to the whole village), and half of this effect was obtained when control was applied only to the 33% of households closest to the village periphery. Peri-domicile cleaning was able to eliminate up to 60% of the vectors, but at the periphery of the village it has a low effect, as it is ineffective against sylvatic insects. The use of lethal traps and the management of house attractiveness provided similar levels of control. However this required either house attractiveness to be null, or ≥5 lethal traps, at least as attractive as houses, to be installed in each household. Conclusion/Significance Insecticide and insect screens used in houses at the periphery of the village can contribute to reduce house infestation in more central untreated zones. However, this beneficial effect remains insufficient to allow for a unique

  17. Targeting the Central Nervous System with Herpes Simplex Virus / Sleeping Beauty Hybrid Amplicon Vectors

    PubMed Central

    de Silva, Suresh; Bowers, William J.

    2014-01-01

    The pursuits of sustainable treatments for diseases and disorders that afflict the central nervous system (CNS) have proven challenging for the field of viral vector-based gene therapy. However, recent advances in viral vector technology coupled with efficient delivery methods have opened up new avenues that show promise at the preclinical testing stage. The development of the Herpes Simplex Virus/Sleeping Beauty (HSV/SB) hybrid vector represents such an advance for devising treatments targeting the CNS with its potential for stably integrating large transgenomic segments of DNA within the genomes of transduced cells. In utero administration of this hybrid vector into the embryonic mouse brain has revealed the capacity for widespread transgene dissemination due to the targeting of a neuronal precursor cell population. This unique feature has provided the means to stably express a transgene throughout the brain for prolonged periods, which is a prerequisite for the treatment of progressive CNS disorders. In this review we provide a comprehensive breakdown of the characteristics of the HSV/SB vector system and how it can be efficiently employed in the derivation of CNS-targeted gene therapeutic strategies. PMID:21711226

  18. Gene delivery to adipose tissue using transcriptionally targeted rAAV8 vectors.

    PubMed

    Uhrig-Schmidt, Silke; Geiger, Matthias; Luippold, Gerd; Birk, Gerald; Mennerich, Detlev; Neubauer, Heike; Grimm, Dirk; Wolfrum, Christian; Kreuz, Sebastian

    2014-01-01

    In recent years, the increasing prevalence of obesity and obesity-related co-morbidities fostered intensive research in the field of adipose tissue biology. To further unravel molecular mechanisms of adipose tissue function, genetic tools enabling functional studies in vitro and in vivo are essential. While the use of transgenic animals is well established, attempts using viral and non-viral vectors to genetically modify adipocytes in vivo are rare. Therefore, we here characterized recombinant Adeno-associated virus (rAAV) vectors regarding their potency as gene transfer vehicles for adipose tissue. Our results demonstrate that a single dose of systemically applied rAAV8-CMV-eGFP can give rise to remarkable transgene expression in murine adipose tissues. Upon transcriptional targeting of the rAAV8 vector to adipocytes using a 2.2 kb fragment of the murine adiponectin (mAP2.2) promoter, eGFP expression was significantly decreased in off-target tissues while efficient transduction was maintained in subcutaneous and visceral fat depots. Moreover, rAAV8-mAP2.2-mediated expression of perilipin A - a lipid-droplet-associated protein - resulted in significant changes in metabolic parameters only three weeks post vector administration. Taken together, our findings indicate that rAAV vector technology is applicable as a flexible tool to genetically modify adipocytes for functional proof-of-concept studies and the assessment of putative therapeutic targets in vivo.

  19. The myeloid-binding peptide adenoviral vector enables multi-organ vascular endothelial gene targeting.

    PubMed

    Lu, Zhi Hong; Kaliberov, Sergey; Zhang, Jingzhu; Muz, Barbara; Azab, Abdel K; Sohn, Rebecca E; Kaliberova, Lyudmila; Du, Yingqiu; Curiel, David T; Arbeit, Jeffrey M

    2014-08-01

    Vascular endothelial cells (ECs) are ideal gene therapy targets as they provide widespread tissue access and are the first contact surfaces following intravenous vector administration. Human recombinant adenovirus serotype 5 (Ad5) is the most frequently used gene transfer system because of its appreciable transgene payload capacity and lack of somatic mutation risk. However, standard Ad5 vectors predominantly transduce liver but not the vasculature following intravenous administration. We recently developed an Ad5 vector with a myeloid cell-binding peptide (MBP) incorporated into the knob-deleted, T4 fibritin chimeric fiber (Ad.MBP). This vector was shown to transduce pulmonary ECs presumably via a vector handoff mechanism. Here we tested the body-wide tropism of the Ad.MBP vector, its myeloid cell necessity, and vector-EC expression dose response. Using comprehensive multi-organ co-immunofluorescence analysis, we discovered that Ad.MBP produced widespread EC transduction in the lung, heart, kidney, skeletal muscle, pancreas, small bowel, and brain. Surprisingly, Ad.MBP retained hepatocyte tropism albeit at a reduced frequency compared with the standard Ad5. While binding specifically to myeloid cells ex vivo, multi-organ Ad.MBP expression was not dependent on circulating monocytes or macrophages. Ad.MBP dose de-escalation maintained full lung-targeting capacity but drastically reduced transgene expression in other organs. Swapping the EC-specific ROBO4 for the CMV promoter/enhancer abrogated hepatocyte expression but also reduced gene expression in other organs. Collectively, our multilevel targeting strategy could enable therapeutic biological production in previously inaccessible organs that pertain to the most debilitating or lethal human diseases.

  20. Efficient and Targeted Transduction of Nonhuman Primate Liver With Systemically Delivered Optimized AAV3B Vectors.

    PubMed

    Li, Shaoyong; Ling, Chen; Zhong, Li; Li, Mengxin; Su, Qin; He, Ran; Tang, Qiushi; Greiner, Dale L; Shultz, Leonard D; Brehm, Michael A; Flotte, Terence R; Mueller, Christian; Srivastava, Arun; Gao, Guangping

    2015-12-01

    Recombinant adeno-associated virus serotype 3B (rAAV3B) can transduce cultured human liver cancer cells and primary human hepatocytes efficiently. Serine (S)- and threonine (T)-directed capsid modifications further augment its transduction efficiency. Systemically delivered capsid-optimized rAAV3B vectors can specifically target cancer cells in a human liver cancer xenograft model, suggesting their potential use for human liver-directed gene therapy. Here, we compared transduction efficiencies of AAV3B and AAV8 vectors in cultured primary human hepatocytes and cancer cells as well as in human and mouse hepatocytes in a human liver xenograft NSG-PiZ mouse model. We also examined the safety and transduction efficacy of wild-type (WT) and capsid-optimized rAAV3B in the livers of nonhuman primates (NHPs). Intravenously delivered S663V+T492V (ST)-modified self-complementary (sc) AAV3B-EGFP vectors led to liver-targeted robust enhanced green fluorescence protein (EGFP) expression in NHPs without apparent hepatotoxicity. Intravenous injections of both WT and ST-modified rAAV3B.ST-rhCG vectors also generated stable super-physiological levels of rhesus chorionic gonadotropin (rhCG) in NHPs. The vector genome predominantly targeted the liver. Clinical chemistry and histopathology examinations showed no apparent vector-related toxicity. Our studies should be important and informative for clinical development of optimized AAV3B vectors for human liver-directed gene therapy.

  1. Selective targeting of human cells by a chimeric adenovirus vector containing a modified fiber protein.

    PubMed Central

    Stevenson, S C; Rollence, M; Marshall-Neff, J; McClelland, A

    1997-01-01

    The adenovirus fiber protein is responsible for attachment of the virion to unidentified cell surface receptors. There are at least two distinct adenovirus fiber receptors which interact with the group B (Ad3) and group C (Ad5) adenoviruses. We have previously shown by using expressed adenovirus fiber proteins that it is possible to change the specificity of the fiber protein by exchanging the head domain with another serotype which recognizes a different receptor (S. C. Stevenson et al., J. Virol. 69:2850-2857, 1995). A chimeric fiber cDNA containing the Ad3 fiber head domain fused to the Ad5 fiber tail and shaft was incorporated into the genome of an adenovirus vector with E1 and E3 deleted encoding beta-galactosidase to generate Av9LacZ4, an adenovirus particle which contains a chimeric fiber protein. Western blot analysis of the chimeric fiber vector confirmed expression of the chimeric fiber protein and its association with the adenovirus capsid. Transduction experiments with fiber protein competitors demonstrated the altered receptor tropism of the chimeric fiber vector compared to that of the parental Av1LacZ4 vector. Transduction of a panel of human cell lines with the chimeric and parental vectors provided evidence for a different cellular distribution of the Ad5 and Ad3 receptors. Three cell lines (THP-1, MRC-5, and FaDu) were more efficiently transduced by the vector containing the Ad3 fiber head than by the Ad5 fiber vector. In contrast, human coronary artery endothelial cells were transduced more readily with the vector containing the Ad5 fiber than with the chimeric fiber vector. HeLa and human umbilical vein endothelial cells were transduced at equivalent levels compared with human diploid fibroblasts, which were refractory to transduction with both vectors. These results provide evidence for the differential expression of the Ad5 and Ad3 receptors on human cell lines derived from clinically relevant target tissues. Furthermore, we show that exchange

  2. Dendritic cell based PSMA immunotherapy for prostate cancer using a CD40-targeted adenovirus vector.

    PubMed

    Williams, Briana Jill; Bhatia, Shilpa; Adams, Lisa K; Boling, Susan; Carroll, Jennifer L; Li, Xiao-Lin; Rogers, Donna L; Korokhov, Nikolay; Kovesdi, Imre; Pereboev, Alexander V; Curiel, David T; Mathis, J Michael

    2012-01-01

    Human prostate tumor vaccine and gene therapy trials using ex vivo methods to prime dendritic cells (DCs) with prostate specific membrane antigen (PSMA) have been somewhat successful, but to date the lengthy ex vivo manipulation of DCs has limited the widespread clinical utility of this approach. Our goal was to improve upon cancer vaccination with tumor antigens by delivering PSMA via a CD40-targeted adenovirus vector directly to DCs as an efficient means for activation and antigen presentation to T-cells. To test this approach, we developed a mouse model of prostate cancer by generating clonal derivatives of the mouse RM-1 prostate cancer cell line expressing human PSMA (RM-1-PSMA cells). To maximize antigen presentation in target cells, both MHC class I and TAP protein expression was induced in RM-1 cells by transduction with an Ad vector expressing interferon-gamma (Ad5-IFNγ). Administering DCs infected ex vivo with CD40-targeted Ad5-huPSMA, as well as direct intraperitoneal injection of the vector, resulted in high levels of tumor-specific CTL responses against RM-1-PSMA cells pretreated with Ad5-IFNγ as target cells. CD40 targeting significantly improved the therapeutic antitumor efficacy of Ad5-huPSMA encoding PSMA when combined with Ad5-IFNγ in the RM-1-PSMA model. These results suggest that a CD-targeted adenovirus delivering PSMA may be effective clinically for prostate cancer immunotherapy.

  3. Dendritic Cell Based PSMA Immunotherapy for Prostate Cancer Using a CD40-Targeted Adenovirus Vector

    PubMed Central

    Williams, Briana Jill; Bhatia, Shilpa; Adams, Lisa K.; Boling, Susan; Carroll, Jennifer L.; Li, Xiao-Lin; Rogers, Donna L.; Korokhov, Nikolay; Kovesdi, Imre; Pereboev, Alexander V.; Curiel, David T.; Mathis, J. Michael

    2012-01-01

    Human prostate tumor vaccine and gene therapy trials using ex vivo methods to prime dendritic cells (DCs) with prostate specific membrane antigen (PSMA) have been somewhat successful, but to date the lengthy ex vivo manipulation of DCs has limited the widespread clinical utility of this approach. Our goal was to improve upon cancer vaccination with tumor antigens by delivering PSMA via a CD40-targeted adenovirus vector directly to DCs as an efficient means for activation and antigen presentation to T-cells. To test this approach, we developed a mouse model of prostate cancer by generating clonal derivatives of the mouse RM-1 prostate cancer cell line expressing human PSMA (RM-1-PSMA cells). To maximize antigen presentation in target cells, both MHC class I and TAP protein expression was induced in RM-1 cells by transduction with an Ad vector expressing interferon-gamma (Ad5-IFNγ). Administering DCs infected ex vivo with CD40-targeted Ad5-huPSMA, as well as direct intraperitoneal injection of the vector, resulted in high levels of tumor-specific CTL responses against RM-1-PSMA cells pretreated with Ad5-IFNγ as target cells. CD40 targeting significantly improved the therapeutic antitumor efficacy of Ad5-huPSMA encoding PSMA when combined with Ad5-IFNγ in the RM-1-PSMA model. These results suggest that a CD-targeted adenovirus delivering PSMA may be effective clinically for prostate cancer immunotherapy. PMID:23056548

  4. Targeting metastatic cancer from the inside: a new generation of targeted gene delivery vectors enables personalized cancer vaccination in situ.

    PubMed

    Gordon, Erlinda M; Levy, John P; Reed, Rebecca A; Petchpud, W Nina; Liu, Liqiong; Wendler, Carlan B; Hall, Frederick L

    2008-10-01

    The advent of pathotropic (disease-seeking) targeting technologies, combined with advanced gene delivery vectors, provides a unique opportunity for the systemic delivery of immunomodulatory cytokine genes to remote sites of cancer metastasis. When injected intravenously, such pathotropic nanoparticles seek out and accumulate selectively at sites of tumor invasion and neo-angiogenesis, resulting in enhanced gene delivery, and thus cytokine production, within the tumor nodules. Used in conjunction with a primary tumoricidal agent (e.g., Rexin-G) that exposes tumor neoantigens, the tumor-targeted immunotherapy vector is intended to promote the recruitment and activation of host immune cells into the metastastic site(s), thereby initiating cancer immunization in situ. In this study, we examine the feasibility of cytokine gene delivery to cancerous lesions in vivo using intravenously administered pathotropically targeted nanoparticles bearing the gene encoding granulocyte/macrophage colony-stimulating factor (GM-CSF; i.e., Reximmune-C). In vitro, transduction of target cancer cells with Reximmune-C resulted in the quantitative production of bioactive and immunoreactive GM-CSF protein. In tumor-bearing nude mice, intravenous infusions of Reximmune-C-induced GM-CSF production by transduced cancer cells and paracrine secretion of the cytokine within the tumor nodules, which promoted the recruitment of host mononuclear cells, including CD40+ B cells and CD86+ dendritic cells, into the tumors. With the first proofs of principle established in preclinical studies, we generated an optimized vector configuration for use in advanced clinical trial designs, and extended the feasibility studies to the clinic. Targeted delivery and localized expression of the GM-CSF transgene was confirmed in a patient with metastatic cancer, as was the recruitment of significant tumor-infiltrating lymphocytes (TILs). Taken together, these studies provide the first demonstrations of cytokine gene

  5. Hypoxia- and radiation-inducible, breast cell-specific targeting of retroviral vectors

    SciTech Connect

    Lipnik, Karoline; Greco, Olga; Scott, Simon; Knapp, Elzbieta; Mayrhofer, Elisabeth; Rosenfellner, Doris; Guenzburg, Walter H.; Salmons, Brian; Hohenadl, Christine . E-mail: christine.hohenadl@vu-wien.ac.at

    2006-05-25

    To facilitate a more efficient radiation and chemotherapy of mammary tumours, synthetic enhancer elements responsive to hypoxia and ionizing radiation were coupled to the mammary-specific minimal promoter of the murine whey acidic protein (WAP) encoding gene. The modified WAP promoter was introduced into a retroviral promoter conversion (ProCon) vector. Expression of a transduced reporter gene in response to hypoxia and radiation was analysed in stably infected mammary cancer cell lines and an up to 9-fold increase in gene expression demonstrated in comparison to the respective basic vector. Expression analyses in vitro, moreover, demonstrated a widely preserved mammary cell-specific promoter activity. For in vivo analyses, xenograft tumours consisting of infected human mammary adenocarcinoma cells were established in SCID/beige mice. Immunohistochemical analyses demonstrated a hypoxia-specific, markedly increased WAP promoter-driven expression in these tumours. Thus, this retroviral vector will facilitate a targeted gene therapeutic approach exploiting the unique environmental condition in solid tumours.

  6. A novel promoterless gene targeting vector to efficiently disrupt PRNP gene in cattle.

    PubMed

    Wang, Shaohua; Zhang, Kun; Ding, Fangrong; Zhao, Rui; Li, Song; Li, Rong; Xu, Lingling; Song, Chi; Dai, Yunping; Li, Ning

    2013-02-20

    The PRNP gene encodes a cellular protein named prion, whose misfolded form has been implicated in a number of neuropathic diseases in mammals such as the Bovine Spongiform Encephalopathy (BSE) in cattle. BSE has brought devastating impact on the world economy and human health. Recently, several groups have performed the gene targeting strategy to disrupt the PRNP gene in bovine fibroblast cells and produce BSE-resistant cattle by somatic cell nuclear transfer (SCNT). However, the enrichment efficiency of the gene targeting vector was low. Here, we constructed a novel promoterless gene targeting vector to sequentially disrupt the PRNP gene in bovine fibroblast cells and generate gene targeted cattle by SCNT. The enrichment efficiency of the novel vector was 100% and 60%, respectively. After nuclear transfer, no significant difference was found in the rate of cleavage and blastocyst formation between the knockout and wild type cloned embryos. One PRNP⁺/⁻ calf was born with no obvious abnormal development by now. Fusion RT-PCR and real-time PCR showed one allele of the PRNP gene was functionally disrupted, and the mRNA expression reduced dramatically in the PRNP⁺/⁻ cattle. The reconstituted PRNP⁻/⁻ embryos showed double alleles disruption, and no difference in the rate of cleavage and blastocyst formation. Copyright © 2012 Elsevier B.V. All rights reserved.

  7. Development of a replication-competent lentivirus assay for dendritic cell-targeting lentiviral vectors

    PubMed Central

    Farley, Daniel C; McCloskey, Laura; Thorne, Barbara A; Tareen, Semih U; Nicolai, Christopher J; Campbell, David J; Bannister, Richard; Stewart, Hannah J; Pearson, Laura JE; Moyer, Bentley J; Robbins, Scott H; Zielinski, Leah; Kim, Tae; Radcliffe, Pippa A; Mitrophanous, Kyriacos A; Gombotz, Wayne R; Miskin, James E; Kelley-Clarke, Brenna

    2015-01-01

    It is a current regulatory requirement to demonstrate absence of detectable replication-competent lentivirus (RCL) in lentiviral vector products prior to use in clinical trials. Immune Design previously described an HIV-1-based integration-deficient lentiviral vector for use in cancer immunotherapy (VP02). VP02 is enveloped with E1001, a modified Sindbis virus glycoprotein which targets dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) expressed on dendritic cells in vivo. Vector enveloped with E1001 does not transduce T-cell lines used in standard HIV-1-based RCL assays, making current RCL testing formats unsuitable for testing VP02. We therefore developed a novel assay to test for RCL in clinical lots of VP02. This assay, which utilizes a murine leukemia positive control virus and a 293F cell line expressing the E1001 receptor DC-SIGN, meets a series of evaluation criteria defined in collaboration with US regulatory authorities and demonstrates the ability of the assay format to amplify and detect a hypothetical RCL derived from VP02 vector components. This assay was qualified and used to test six independent GMP production lots of VP02, in which no RCL was detected. We propose that the evaluation criteria used to rationally design this novel method should be considered when developing an RCL assay for any lentiviral vector. PMID:26029728

  8. A biocleavable pullulan-based vector via ATRP for liver cell-targeting gene delivery.

    PubMed

    Yang, Xin-Chao; Niu, Yan-Lan; Zhao, Na-Na; Mao, Chun; Xu, Fu-Jian

    2014-04-01

    Pullulan due to its specificity for liver has been widely exploited for biomedical applications. In this work, a tailor-made biocleavable pullulan-based gene vector (PuPGEA) with good hemocompatibility was successfully proposed via atom transfer radical polymerization (ATRP) for efficient liver cell-targeting gene delivery. A two-step method involving the reaction of hydroxyl groups of pullulan with cystamine was developed to introduce reduction-sensitive disulfide-linked initiation sites of ATRP onto pullulan. The poly(glycidyl methacrylate) (PGMA) side chains prepared subsequently via ATRP were functionalized with ethanolamine (EA) to produce the resultant biocleavable comb-shaped PuPGEA vectors consisting of nonionic pullulan backbones and disulfide-linked cationic EA-functionalized PGMA (PGEA) side chains with plentiful secondary amine and nonionic hydroxyl units. The cationic PGEA side chains can be readily cleavable from the pullulan backbones of PuPGEA under reducible conditions. Due to the liver targeting performance of pullulan backbones, such PuPGEA vectors exhibited much higher gene transfection efficiency and cellular uptake rates in HepG2 cell lines than in Hella cell lines. In addition, in vitro transfection efficiency and uptake mechanism of polyplex in HepG2 cells were evaluated in the presence of different endocytosis inhibitors, indicating that the asialoglycoprotein receptor was involved in transfection process of hepatocytes. More importantly, in comparison with gold standard polyethylenimine (PEI, ∼25 kDa), PuPGEA vectors possessed excellent hemocompatibility without causing undesirable hemolysis. Properly grafting short bioreducible PGEA polycation side chains from a liver cell-targeting pullulan backbone is an effective means to produce new hemocompatible polysaccharide-based gene delivery vectors. Copyright © 2014 Elsevier Ltd. All rights reserved.

  9. Embryonic stem cell gene targeting using bacteriophage lambda vectors generated by phage-plasmid recombination.

    PubMed Central

    Tsuzuki, T; Rancourt, D E

    1998-01-01

    Targeted mutagenesis is an extremely useful experimental approach in molecular medicine, allowing the generation of specialized animals that are mutant for any gene of interest. Currently the rate determining step in any gene targeting experiment is construction of the targeting vector (TV). In order to streamline gene targeting methods and avoid problems encountered with plasmid TVs, we describe the direct application of lambda phage in targeted mutagenesis. The recombination-proficient phage vector lambda2TK permits generation of TVs by conventional restriction-ligation or recombination-mediated methods. The resulting lambdaTV DNA can then be cleaved with restriction endonucleases to release the bacteriophage arms and can subsequently be electroporated directly into ES cells to yield gene targets. We demonstrate that in vivo phage-plasmid recombination can be used to introduce neo and lacZ - neo mutations into precise positions within a lambda2TK subclone via double crossover recombination. We describe two methods for eliminating single crossover recombinants, spi selection and size restriction, both of which result in phage TVs bearing double crossover insertions. Thus TVs can be easily and quickly generated in bacteriophage without plasmid subcloning and with little genomic sequence or restriction site information. PMID:9461458

  10. Subcloning plus insertion (SPI)--a novel recombineering method for the rapid construction of gene targeting vectors.

    PubMed

    Reddy, Thimma R; Kelsall, Emma J; Fevat, Léna M S; Munson, Sarah E; Cowley, Shaun M

    2015-01-08

    Gene targeting refers to the precise modification of a genetic locus using homologous recombination. The generation of novel cell lines and transgenic mouse models using this method necessitates the construction of a 'targeting' vector, which contains homologous DNA sequences to the target gene, and has for many years been a limiting step in the process. Vector construction can be performed in vivo in Escherichia coli cells using homologous recombination mediated by phage recombinases using a technique termed recombineering. Recombineering is the preferred technique to subclone the long homology sequences (>4 kb) and various targeting elements including selection markers that are required to mediate efficient allelic exchange between a targeting vector and its cognate genomic locus. Typical recombineering protocols follow an iterative scheme of step-wise integration of the targeting elements and require intermediate purification and transformation steps. Here, we present a novel recombineering methodology of vector assembly using a multiplex approach. Plasmid gap repair is performed by the simultaneous capture of genomic sequence from mouse Bacterial Artificial Chromosome libraries and the insertion of dual bacterial and mammalian selection markers. This subcloning plus insertion method is highly efficient and yields a majority of correct recombinants. We present data for the construction of different types of conditional gene knockout, or knock-in, vectors and BAC reporter vectors that have been constructed using this method. SPI vector construction greatly extends the repertoire of the recombineering toolbox and provides a simple, rapid and cost-effective method of constructing these highly complex vectors.

  11. [Construction and identification of small interfering RNA expression vector targeting ATF-2 gene].

    PubMed

    Mao, Wei-wei; Xiong, Peng; Han, Feng; Hu, Zhi-jian

    2012-09-01

    To construct an eukaryotic expression vector for RNA interference targeting activating transcription factor 2 (ATF-2) gene, and explore its effect on proliferation and apoptosis of HepG2 cells. Two complementary oligonucleotides were synthesized based on ATF-2 mRNA sequence. The annealed fragment was inserted into the vector PBA-siU6. The recombinant plasmid PBA-siATF-2 was confirmed by DNA sequencing and transfected into HepG2 cells mediated by liposome. After transfection, ATF-2 protein was detected by Western blotting. The cellular growth activity and apoptosis rate were measured by MTT assay and flow cytometry, respectively. Recombinant plasmid expressing siRNA targeting ATF-2 gene was confirmed by DNA sequencing. Plasmid transfection down-regulated the level of ATF-2 protein in HepG2 cells, which blocked cellular growth and induced cell apoptosis. The eukaryotic expression vector for RNA interference targeting ATF-2 gene was constructed successfully, which inhibits HepG2 cell proliferation and induces cell apoptosis.

  12. A hierarchical classifier using new support vector machines for automatic target recognition.

    PubMed

    Casasent, David; Wang, Yu-Chiang

    2005-01-01

    A binary hierarchical classifier is proposed for automatic target recognition. We also require rejection of non-object (non-target) inputs, which are not seen during training or validation, thus producing a very difficult problem. The SVRDM (support vector representation and discrimination machine) classifier is used at each node in the hierarchy, since it offers good generalization and rejection ability. Using this hierarchical SVRDM classifier with magnitude Fourier transform (|FT|) features, which provide shift-invariance, initial test results on infra-red (IR) data are excellent.

  13. A protocol for construction of gene targeting vectors and generation of homologous recombinant embryonic stem cells.

    PubMed

    Bouabe, Hicham; Okkenhaug, Klaus

    2013-01-01

    The completion of human and mouse genome sequencing has confronted us with huge amount of data sequences that certainly need decades and many generations of scientists to be reasonably interpreted and assigned to physiological functions, and subsequently fruitfully translated into medical application. A means to assess the function of genes provides gene targeting in mouse embryonic stem cells (ESCs) that enables to introduce site-specific modifications in the mouse genome, and analyze their physiological consequences. Gene targeting enables almost any type of genetic modifications of interest, ranging from gene insertion (e.g., insertion of human-specific genes or reporter genes), gene disruption, point mutations, and short- and long-range deletions, inversions. Site-specific modification into the genome of ESCs can be reached by homologous recombination using targeting vectors. Here, we describe a protocol to generate targeting constructs and homologous recombinant ESCs.

  14. A targeted change-detection procedure by combining change vector analysis and post-classification approach

    NASA Astrophysics Data System (ADS)

    Ye, Su; Chen, Dongmei; Yu, Jie

    2016-04-01

    In remote sensing, conventional supervised change-detection methods usually require effective training data for multiple change types. This paper introduces a more flexible and efficient procedure that seeks to identify only the changes that users are interested in, here after referred to as ;targeted change detection;. Based on a one-class classifier ;Support Vector Domain Description (SVDD);, a novel algorithm named ;Three-layer SVDD Fusion (TLSF); is developed specially for targeted change detection. The proposed algorithm combines one-class classification generated from change vector maps, as well as before- and after-change images in order to get a more reliable detecting result. In addition, this paper introduces a detailed workflow for implementing this algorithm. This workflow has been applied to two case studies with different practical monitoring objectives: urban expansion and forest fire assessment. The experiment results of these two case studies show that the overall accuracy of our proposed algorithm is superior (Kappa statistics are 86.3% and 87.8% for Case 1 and 2, respectively), compared to applying SVDD to change vector analysis and post-classification comparison.

  15. Magnetically vectored platforms for the targeted delivery of therapeutics to tumors: history and current status.

    PubMed

    Seeney, Charles; Ojwang, Joshua O; Weiss, Ronald D; Klostergaard, Jim

    2012-02-01

    Superparamagnetic iron oxide nanoparticles (SPIONs) are being developed as vehicles for the selective targeting of therapeutics and bioactive compounds. Presented herein is a brief review of the history of approaches to magnetic-based drug delivery platforms, leading to current concepts of magnetically vectored therapeutics via functionalized SPION-prodrugs. With this background, recent experimental results are discussed that demonstrate the use of shaped external magnetic field gradients, generated by designed configurations of permanent magnets, to drive the concentration/accumulation of modified SPION-prodrug constructs at a tumor site, followed by tumor extravasation and activation of the prodrug within the tumor microenvironment. In order to successfully translate this approach to clinical application, one of the key requirements is the ability to magnetically drive ('vector') the SPION to human-scale tumor settings. In this review, various configurations of permanent magnets are described and models are presented that demonstrate that magnetic field gradients can potentially be focused and extended to lengths of several inches in vivo. This modification thereby increases the range of the delivery platform, and offers the potential for the treatment of visceral as well as superficial tumors and for translation from preclinical animal tumor models to clinical settings. The methodology of magnetically vectored prodrug therapeutics, as a means for selective localized targeting of tumor tissue, and minimizing harm to normal tissue, has the additional advantage of raising the therapeutic index compared with that of free drugs, thus, offering great potential as a cancer treatment modality.

  16. Tropism-Modification Strategies for Targeted Gene Delivery Using Adenoviral Vectors

    PubMed Central

    Coughlan, Lynda; Alba, Raul; Parker, Alan L.; Bradshaw, Angela C.; McNeish, Iain A.; Nicklin, Stuart A.; Baker, Andrew H.

    2010-01-01

    Achieving high efficiency, targeted gene delivery with adenoviral vectors is a long-standing goal in the field of clinical gene therapy. To achieve this, platform vectors must combine efficient retargeting strategies with detargeting modifications to ablate native receptor binding (i.e. CAR/integrins/heparan sulfate proteoglycans) and “bridging” interactions. “Bridging” interactions refer to coagulation factor binding, namely coagulation factor X (FX), which bridges hepatocyte transduction in vivo through engagement with surface expressed heparan sulfate proteoglycans (HSPGs). These interactions can contribute to the off-target sequestration of Ad5 in the liver and its characteristic dose-limiting hepatotoxicity, thereby significantly limiting the in vivo targeting efficiency and clinical potential of Ad5-based therapeutics. To date, various approaches to retargeting adenoviruses (Ad) have been described. These include genetic modification strategies to incorporate peptide ligands (within fiber knob domain, fiber shaft, penton base, pIX or hexon), pseudotyping of capsid proteins to include whole fiber substitutions or fiber knob chimeras, pseudotyping with non-human Ad species or with capsid proteins derived from other viral families, hexon hypervariable region (HVR) substitutions and adapter-based conjugation/crosslinking of scFv, growth factors or monoclonal antibodies directed against surface-expressed target antigens. In order to maximize retargeting, strategies which permit detargeting from undesirable interactions between the Ad capsid and components of the circulatory system (e.g. coagulation factors, erythrocytes, pre-existing neutralizing antibodies), can be employed simultaneously. Detargeting can be achieved by genetic ablation of native receptor-binding determinants, ablation of “bridging interactions” such as those which occur between the hexon of Ad5 and coagulation factor X (FX), or alternatively, through the use of polymer-coated

  17. Chimeric adeno-associated virus and bacteriophage: a potential targeted gene therapy vector for malignant glioma.

    PubMed

    Asavarut, Paladd; O'Neill, Kevin; Syed, Nelofer; Hajitou, Amin

    2014-01-01

    The incipient development of gene therapy for cancer has fuelled its progression from bench to bedside in mere decades. Of all malignancies that exist, gliomas are the largest class of brain tumors, and are renowned for their aggressiveness and resistance to therapy. In order for gene therapy to achieve clinical success, a multitude of barriers ranging from glioma tumor physiology to vector biology must be overcome. Many viral gene delivery systems have been subjected to clinical investigation; however, with highly limited success. In this review, the current progress and challenges of gene therapy for malignant glioma are discussed. Moreover, we highlight the hybrid adeno-associated virus and bacteriophage vector as a potential candidate for targeted gene delivery to brain tumors.

  18. Tumor-specific suicide gene therapy for hepatocellular carcinoma by transcriptionally targeted retroviral replicating vectors.

    PubMed

    Lai, Y-H; Lin, C-C; Chen, S-H; Tai, C-K

    2015-02-01

    Replicating virus vectors are attractive tools for anticancer gene therapy, but the potential for adverse events due to uncontrolled spread of the vectors has been a major concern. To design a tumor-specific retroviral replicating vector (RRV), we replaced the U3 region of the RRV ACE-GFP with a regulatory sequence consisting of the hepatitis B virus enhancer II (EII) and human α-fetoprotein (AFP) core promoter to produce ACE-GFP-EIIAFP, a hepatocellular carcinoma (HCC)-targeting RRV. Similar to ACE-GFP, ACE-GFP-EIIAFP exhibited robust green fluorescent protein (GFP) expression in HCC cells and, most importantly, it exhibited HCC-specific replication and did not replicate in non-HCC tumor cells or normal liver cells. We sequenced the promoter region of ACE-GFP-EIIAFP collected from serial infection cycles to examine the genomic stability of the vector during its replicative spread, and found that the vector could retain the hybrid promoter in the genome for at least six infection cycles. In vitro studies revealed that ACE-CD-EIIAFP and ACE-PNP-EIIAFP, which express the yeast cytosine deaminase and Escherichia coli purine nucleoside phosphorylase, respectively, exert a highly potent cytotoxic effect on HCC cells in the presence of their respective prodrugs. In vivo, ACE-CD-EIIAFP-mediated suicide gene therapy efficiently suppressed HCC tumor growth and no detectable RRV signal was observed in extratumoral tissues. These results suggest that the tumor-specific, suicide-gene-encoding RRV may fulfill the promise of retroviral gene therapy for cancer.

  19. A technique for measurement of vector and tensor polarization in solid spin one polarized targets

    SciTech Connect

    Kielhorn, W.F.

    1991-06-01

    Vector and tensor polarizations are explicitly defined and used to characterize the polarization states of spin one polarized targets, and a technique for extracting these polarizations from nuclear magnetic resonance (NMR) data is developed. This technique is independent of assumptions about spin temperature, but assumes the target's crystal structure induces a quadrupole interaction with the spin one particles. Analysis of the NMR signals involves a computer curve fitting algorithm implemented with a fast Fourier transform method which speeds and simplifies curve fitting algorithms used previously. For accurate curve fitting, the NMR electronic circuit must be modeled by the fitting algorithm. Details of a circuit, its model, and data collected from this circuit are given for a solid deuterated ammonia target. 37 refs., 19 figs., 3 tabs.

  20. Detecting and sorting targeting peptides with neural networks and support vector machines.

    PubMed

    Hawkins, John; Bodén, Mikael

    2006-02-01

    This paper presents a composite multi-layer classifier system for predicting the subcellular localization of proteins based on their amino acid sequence. The work is an extension of our previous predictor PProwler v1.1 which is itself built upon the series of predictors SignalP and TargetP. In this study we outline experiments conducted to improve the classifier design. The major improvement came from using Support Vector machines as a "smart gate" sorting the outputs of several different targeting peptide detection networks. Our final model (PProwler v1.2) gives MCC values of 0.873 for non-plant and 0.849 for plant proteins. The model improves upon the accuracy of our previous subcellular localization predictor (PProwler v1.1) by 2% for plant data (which represents 7.5% improvement upon TargetP).

  1. Lentiviral Vector-based Insertional Mutagenesis Identifies Genes Involved in the Resistance to Targeted Anticancer Therapies

    PubMed Central

    Ranzani, Marco; Annunziato, Stefano; Calabria, Andrea; Brasca, Stefano; Benedicenti, Fabrizio; Gallina, Pierangela; Naldini, Luigi; Montini, Eugenio

    2014-01-01

    The high transduction efficiency of lentiviral vectors in a wide variety of cells makes them an ideal tool for forward genetics screenings addressing issues of cancer research. Although molecular targeted therapies have provided significant advances in tumor treatment, relapses often occur by the expansion of tumor cell clones carrying mutations that confer resistance. Identification of the culprits of anticancer drug resistance is fundamental for the achievement of long-term response. Here, we developed a new lentiviral vector-based insertional mutagenesis screening to identify genes that confer resistance to clinically relevant targeted anticancer therapies. By applying this genome-wide approach to cell lines representing two subtypes of HER2+ breast cancer, we identified 62 candidate lapatinib resistance genes. We validated the top ranking genes, i.e., PIK3CA and PIK3CB, by showing that their forced expression confers resistance to lapatinib in vitro and found that their mutation/overexpression is associated to poor prognosis in human breast tumors. Then, we successfully applied this approach to the identification of erlotinib resistance genes in pancreatic cancer, thus showing the intrinsic versatility of the approach. The acquired knowledge can help identifying combinations of targeted drugs to overcome the occurrence of resistance, thus opening new horizons for more effective treatment of tumors. PMID:25195596

  2. Efficient conditional knockout targeting vector construction using co-selection BAC recombineering (CoSBR)

    PubMed Central

    Newman, Robert J.; Roose-Girma, Merone; Warming, Søren

    2015-01-01

    A simple and efficient strategy for Bacterial Artificial Chromosome (BAC) recombineering based on co-selection is described. We show that it is possible to efficiently modify two positions of a BAC simultaneously by co-transformation of a single-stranded DNA oligo and a double-stranded selection cassette. The use of co-selection BAC recombineering reduces the DNA manipulation needed to make a conditional knockout gene targeting vector to only two steps: a single round of BAC modification followed by a retrieval step. PMID:26089387

  3. Comparison of adenovirus fiber, protein IX, and hexon capsomeres as scaffolds for vector purification and cell targeting

    SciTech Connect

    Campos, Samuel K.; Barry, Michael A. . E-mail: mab@bcm.edu

    2006-06-05

    The direct genetic modification of adenoviral capsid proteins with new ligands is an attractive means to confer targeted tropism to adenoviral vectors. Although several capsid proteins have been reported to tolerate the genetic fusion of foreign peptides and proteins, direct comparison of cell targeting efficiencies through the different capsomeres has been lacking. Likewise, direct comparison of with one or multiple ligands has not been performed due to a lack of capsid-compatible ligands available for retargeting. Here we utilize a panel of metabolically biotinylated Ad vectors to directly compare targeted transduction through the fiber, protein IX, and hexon capsomeres using a variety of biotinylated ligands including antibodies, transferrin, EGF, and cholera toxin B. These results clearly demonstrate that cell targeting with a variety of high affinity receptor-binding ligands is only effective when transduction is redirected through the fiber protein. In contrast, protein IX and hexon-mediated targeting by the same set of ligands failed to mediate robust vector targeting, perhaps due to aberrant trafficking at the cell surface or inside targeted cells. These data suggest that vector targeting by genetic incorporation of high affinity ligands will likely be most efficient through modification of the adenovirus fiber rather than the protein IX and hexon capsomeres. In contrast, single-step monomeric avidin affinity purification of Ad vectors using the metabolic biotinylation system is most effective through capsomeres like protein IX and hexon.

  4. In silico models for predicting vector control chemicals targeting Aedes aegypti.

    PubMed

    Devillers, J; Lagneau, C; Lattes, A; Garrigues, J C; Clémenté, M M; Yébakima, A

    2014-01-01

    Human arboviral diseases have emerged or re-emerged in numerous countries worldwide due to a number of factors including the lack of progress in vaccine development, lack of drugs, insecticide resistance in mosquitoes, climate changes, societal behaviours, and economical constraints. Thus, Aedes aegypti is the main vector of the yellow fever and dengue fever flaviviruses and is also responsible for several recent outbreaks of the chikungunya alphavirus. As for the other mosquito species, the A. aegypti control relies heavily on the use of insecticides. However, because of increasing resistance to the different families of insecticides, reduction of Aedes populations is becoming increasingly difficult. Despite the unquestionable utility of insecticides in fighting mosquito populations, there are very few new insecticides developed and commercialized for vector control. This is because the high cost of the discovery of an insecticide is not counterbalanced by the 'low profitability' of the vector control market. Fortunately, the use of quantitative structure-activity relationship (QSAR) modelling allows the reduction of time and cost in the discovery of new chemical structures potentially active against mosquitoes. In this context, the goal of the present study was to review all the existing QSAR models on A. aegypti. The homology and pharmacophore models were also reviewed. Specific attention was paid to show the variety of targets investigated in Aedes in relation to the physiology and ecology of the mosquito as well as the diversity of the chemical structures which have been proposed, encompassing man-made and natural substances.

  5. Synthesis of Bisethylnorspermine Lipid Prodrug as Gene Delivery Vector Targeting Polyamine Metabolism in Breast Cancer

    PubMed Central

    Dong, Yanmei; Zhu, Yu; Li, Jing; Zhou, Qing-Hui; Wu, Chao; Oupický, David

    2013-01-01

    Progress in the development of nonviral gene delivery vectors continues to be hampered by low transfection activity and toxicity. Here we proposed to develop a lipid prodrug based on a polyamine analogue bisethylnorspermine (BSP) that can function dually as gene delivery vector and, after intracellular degradation, as active anticancer agent targeting dysregulated polyamine metabolism. We synthesized a prodrug of BSP (LS-BSP) capable of intracellular release of BSP using thiolytically sensitive dithiobenzyl carbamate linker. Biodegradability of LS-BSP contributed to decreased toxicity compared with nondegradable control L-BSP. BSP showed a strong synergistic enhancement of cytotoxic activity of TNF-related apoptosis-inducing ligand (TRAIL) in human breast cancer cells. Decreased enhancement of TRAIL activity was observed for LS-BSP when compared with BSP. LS-BSP formed complexes with plasmid DNA and mediated transfection activity comparable to DOTAP and L-BSP. Our results show that BSP-based vectors are promising candidates for combination drug/gene delivery. PMID:22545813

  6. In silico models for predicting vector control chemicals targeting Aedes aegypti

    PubMed Central

    Devillers, J.; Lagneau, C.; Lattes, A.; Garrigues, J.C.; Clémenté, M.M.; Yébakima, A.

    2014-01-01

    Human arboviral diseases have emerged or re-emerged in numerous countries worldwide due to a number of factors including the lack of progress in vaccine development, lack of drugs, insecticide resistance in mosquitoes, climate changes, societal behaviours, and economical constraints. Thus, Aedes aegypti is the main vector of the yellow fever and dengue fever flaviviruses and is also responsible for several recent outbreaks of the chikungunya alphavirus. As for the other mosquito species, the A. aegypti control relies heavily on the use of insecticides. However, because of increasing resistance to the different families of insecticides, reduction of Aedes populations is becoming increasingly difficult. Despite the unquestionable utility of insecticides in fighting mosquito populations, there are very few new insecticides developed and commercialized for vector control. This is because the high cost of the discovery of an insecticide is not counterbalanced by the ‘low profitability’ of the vector control market. Fortunately, the use of quantitative structure–activity relationship (QSAR) modelling allows the reduction of time and cost in the discovery of new chemical structures potentially active against mosquitoes. In this context, the goal of the present study was to review all the existing QSAR models on A. aegypti. The homology and pharmacophore models were also reviewed. Specific attention was paid to show the variety of targets investigated in Aedes in relation to the physiology and ecology of the mosquito as well as the diversity of the chemical structures which have been proposed, encompassing man-made and natural substances. PMID:25275884

  7. Transcellular targeting of fiber- and hexon-modified adenovirus vectors across the brain microvascular endothelial cells in vitro.

    PubMed

    Laakkonen, Johanna P; Engler, Tatjana; Romero, Ignacio A; Weksler, Babette; Couraud, Pierre-Olivier; Kreppel, Florian; Kochanek, Stefan

    2012-01-01

    In central nervous system (CNS)-directed gene therapy, efficient targeting of brain parenchyma through the vascular route is prevented by the endothelium and the epithelium of the blood-brain and the blood-cerebrospinal fluid barriers, respectively. In this study, we evaluated the feasibility of the combined genetic and chemical adenovirus capsid modification technology to enable transcellular delivery of targeted adenovirus (Ad) vectors across the blood-brain barrier (BBB) in vitro models. As a proof-of-principle ligand, maleimide-activated full-length human transferrin (hTf) was covalently attached to cysteine-modified Ad serotype 5 vectors either to its fiber or hexon protein. In transcytosis experiments, hTf-coupled vectors were shown to be redirected across the BBB models, the transcytosis activity of the vectors being dependent on the location of the capsid modification and the in vitro model used. The transduction efficiency of hTf-targeted vectors decreased significantly in confluent, polarized cells, indicating that the intracellular route of the vectors differed between unpolarized and polarized cells. After transcellular delivery the majority of the hTf-modified vectors remained intact and partly capable of gene transfer. Altogether, our results demonstrate that i) covalent attachment of a ligand to Ad capsid can mediate transcellular targeting across the cerebral endothelium in vitro, ii) the attachment site of the ligand influences its transcytosis efficiency and iii) combined genetic/chemical modification of Ad vector can be used as a versatile platform for the development of Ad vectors for transcellular targeting.

  8. Plectin-1 Targeted AAV Vector for the Molecular Imaging of Pancreatic Cancer.

    PubMed

    Konkalmatt, Prasad R; Deng, Defeng; Thomas, Stephanie; Wu, Michael T; Logsdon, Craig D; French, Brent A; Kelly, Kimberly A

    2013-01-01

    Pancreatic ductal adenocarcinoma (PDAC) is highly malignant disease that is the fourth leading cause of cancer-related death in the US. Gene therapy using AAV vectors to selectively deliver genes to PDAC cells is an attractive treatment option for pancreatic cancer. However, most AAV serotypes display a broad spectrum of tissue tropism and none of the existing serotypes specifically target PDAC cells. This study tests the hypothesis that AAV2 can be genetically re-engineered to specifically target PDAC cells by modifying the capsid surface to display a peptide that has previously been shown to bind plectin-1. Toward this end, a Plectin-1 Targeting Peptide (PTP) was inserted into the loop IV region of the AAV2 capsid, and the resulting capsid (AAV-PTP) was used in a series of in vitro and in vivo experiments. In vitro, AAV-PTP was found to target all five human PDAC cell lines tested (PANC-1, MIA PaCa-2, HPAC, MPanc-96, and BxPC-3) preferentially over two non-neoplastic human pancreatic cell lines (human pancreatic ductal epithelial and human pancreatic stellate cells). In vivo, mice bearing subcutaneous tumor xenografts were generated using the PANC-1 cell line. Once tumors reached a size of ∼1-2 mm in diameter, the mice were injected intravenously with luciferase reporter vectors packaged in the either AAV-PTP or wild type AAV2 capsids. Luciferase expression was then monitored by bioluminescence imaging on days 3, 7, and 14 after vector injection. The results indicate that the AAV-PTP capsid displays a 37-fold preference for PANC-1 tumor xenographs over liver and other tissues; whereas the wild type AAV2 capsid displays a complementary preference for liver over tumors and other tissues. Together, these results establish proof-of-principle for the ability of PTP-modified AAV capsids to selectively target gene delivery to PDAC cells in vivo, which opens promising new avenues for the early detection, diagnosis, and treatment of pancreatic cancer.

  9. Targeting cells with single vectors using multiple-feature Boolean logic.

    PubMed

    Fenno, Lief E; Mattis, Joanna; Ramakrishnan, Charu; Hyun, Minsuk; Lee, Soo Yeun; He, Miao; Tucciarone, Jason; Selimbeyoglu, Aslihan; Berndt, Andre; Grosenick, Logan; Zalocusky, Kelly A; Bernstein, Hannah; Swanson, Haley; Perry, Chelsey; Diester, Ilka; Boyce, Frederick M; Bass, Caroline E; Neve, Rachael; Huang, Z Josh; Deisseroth, Karl

    2014-07-01

    Precisely defining the roles of specific cell types is an intriguing frontier in the study of intact biological systems and has stimulated the rapid development of genetically encoded tools for observation and control. However, targeting these tools with adequate specificity remains challenging: most cell types are best defined by the intersection of two or more features such as active promoter elements, location and connectivity. Here we have combined engineered introns with specific recombinases to achieve expression of genetically encoded tools that is conditional upon multiple cell-type features, using Boolean logical operations all governed by a single versatile vector. We used this approach to target intersectionally specified populations of inhibitory interneurons in mammalian hippocampus and neurons of the ventral tegmental area defined by both genetic and wiring properties. This flexible and modular approach may expand the application of genetically encoded interventional and observational tools for intact-systems biology.

  10. A recombineering based approach for high-throughput conditional knockout targeting vector construction

    PubMed Central

    Chan, Waiin; Costantino, Nina; Li, Ruixue; Lee, Song Choon; Su, Qin; Melvin, David; Court, Donald L.; Liu, Pentao

    2007-01-01

    Functional analysis of mammalian genes in vivo is primarily achieved through analysing knockout mice. Now that the sequencing of several mammalian genomes has been completed, understanding functions of all the genes represents the next major challenge in the post-genome era. Generation of knockout mutant mice has currently been achieved by many research groups but only by making individual knockouts, one by one. New technological advances and the refinements of existing technologies are critical for genome-wide targeted mutagenesis in the mouse. We describe here new recombineering reagents and protocols that enable recombineering to be carried out in a 96-well format. Consequently, we are able to construct 96 conditional knockout targeting vectors simultaneously. Our new recombineering system makes it a reality to generate large numbers of precisely engineered DNA constructs for functional genomics studies. PMID:17426124

  11. Construction of a pIX-modified Adenovirus Vector Able to Effectively Bind to Nanoantibodies for Targeting.

    PubMed

    Garas, M N; Tillib, S V; Zubkova, O V; Rogozhin, V N; Ivanova, T I; Vasilev, L A; Logunov, D Yu; Shmarov, M M; Tutykhina, I L; Esmagambetov, I B; Gribova, I Yu; Bandelyuk, A S; Naroditsky, B S; Gintsburg, A L

    2014-04-01

    Current targeting strategies for genetic vectors imply the creation of a specific vector for every targeted receptor, which is time-consuming and expensive. Therefore, the development of a universal vector system whose surface can specifically bind molecules to provide efficient targeting is of particular interest. In this study, we propose a new approach in creating targeted vectors based on the genome of human adenovirus serotype 5 carrying the modified gene of the capsid protein pIX (Ad5-EGFP-pIX-ER): recombinant pseudoadenoviral nanoparticles (RPANs). The surfaces of such RPANs are able to bind properly modified chimeric nanoantibodies that specifically recognize a particular target antigen (carcinoembryonic antigen (CEA)) with high affinity. The efficient binding of nanoantibodies (aCEA-RE) to the RPAN capsid surfaces has been demonstrated by ELISA. The ability of the constructed vector to deliver target genes has been confirmed by experiments with the tumor cell lines A549 and Lim1215 expressing CEA. It has been shown that Ad5-EGFP-pIX-ER carrying aCEA-RE on its surface penetrates into the tumor cell lines A549 and Lim1215 via the CAR-independent pathway three times more efficiently than unmodified RPAN and Ad5-EGFP-pIX-ER without nanoantibodies on the capsid surface. Thus, RPAN Ad5-EGFP-pIX-ER is a universal platform that may be useful for targeted gene delivery in specific cells due to "nanoantibody-modified RPAN" binding.

  12. Construction of a pIX-modified Adenovirus Vector Able to Effectively Bind to Nanoantibodies for Targeting

    PubMed Central

    Garas, M. N.; Tillib, S. V.; Zubkova, O. V.; Rogozhin, V. N.; Ivanova, T. I.; Vasilev, L. A.; Logunov, D. Yu.; Shmarov, M. M.; Tutykhina, I. L.; Esmagambetov, I. B.; Gribova, I. Yu.; Bandelyuk, A. S.; Naroditsky, B. S.; Gintsburg, A. L.

    2014-01-01

    Current targeting strategies for genetic vectors imply the creation of a specific vector for every targeted receptor, which is time-consuming and expensive. Therefore, the development of a universal vector system whose surface can specifically bind molecules to provide efficient targeting is of particular interest. In this study, we propose a new approach in creating targeted vectors based on the genome of human adenovirus serotype 5 carrying the modified gene of the capsid protein pIX (Ad5-EGFP-pIX-ER): recombinant pseudoadenoviral nanoparticles (RPANs). The surfaces of such RPANs are able to bind properly modified chimeric nanoantibodies that specifically recognize a particular target antigen (carcinoembryonic antigen (CEA)) with high affinity. The efficient binding of nanoantibodies (aCEA-RE) to the RPAN capsid surfaces has been demonstrated by ELISA. The ability of the constructed vector to deliver target genes has been confirmed by experiments with the tumor cell lines A549 and Lim1215 expressing CEA. It has been shown that Ad5-EGFP-pIX-ER carrying aCEA-RE on its surface penetrates into the tumor cell lines A549 and Lim1215 via the CAR-independent pathway three times more efficiently than unmodified RPAN and Ad5-EGFP-pIX-ER without nanoantibodies on the capsid surface. Thus, RPAN Ad5-EGFP-pIX-ER is a universal platform that may be useful for targeted gene delivery in specific cells due to “nanoantibody–modified RPAN” binding. PMID:25093116

  13. Adeno-associated virus Rep-mediated targeting of integrase-defective retroviral vector DNA circles into human chromosome 19

    SciTech Connect

    Huang, Shuohao; Kawabe, Yoshinori; Ito, Akira; Kamihira, Masamichi

    2012-01-06

    Highlights: Black-Right-Pointing-Pointer Adeno-associated virus (AAV) is capable of targeted integration in human cells. Black-Right-Pointing-Pointer Integrase-defective retroviral vector (IDRV) enables a circular DNA delivery. Black-Right-Pointing-Pointer A targeted integration system of IDRV DNA using the AAV integration mechanism. Black-Right-Pointing-Pointer Targeted IDRV integration ameliorates the safety concerns for retroviral vectors. -- Abstract: Retroviral vectors have been employed in clinical trials for gene therapy owing to their relative large packaging capacity, alterable cell tropism, and chromosomal integration for stable transgene expression. However, uncontrollable integrations of transgenes are likely to cause safety issues, such as insertional mutagenesis. A targeted transgene integration system for retroviral vectors, therefore, is a straightforward way to address the insertional mutagenesis issue. Adeno-associated virus (AAV) is the only known virus capable of targeted integration in human cells. In the presence of AAV Rep proteins, plasmids possessing the p5 integration efficiency element (p5IEE) can be integrated into the AAV integration site (AAVS1) in the human genome. In this report, we describe a system that can target the circular DNA derived from non-integrating retroviral vectors to the AAVS1 site by utilizing the Rep/p5IEE integration mechanism. Our results showed that after G418 selection 30% of collected clones had retroviral DNA targeted at the AAVS1 site.

  14. Characterization of an oncolytic adenovirus vector constructed to target the cMet receptor

    PubMed Central

    Sakr, Hany I; Coleman, David T; Cardelli, James A; Mathis, J Michael

    2015-01-01

    The cMet receptor is a homodimer with tyrosine kinase activity. Upon stimulation with its ligand, hepatocyte growth factor (HGF), the receptor mediates wide physiologic actions. The HGF-cMet signaling pathway is dysregulated in many cancers, which makes cMet an important target for novel therapeutic interventions. Oncolytic adenoviruses (Ads) have been used for the past three decades as a promising therapeutic approach for a wide array of neoplastic diseases. To date, achieving cancer-specific replication of oncolytic Ads has been accomplished by either viral genome deletions or by incorporating tumor selective promoters. To achieve novel specificity of oncolytic Ad infection of cancer cells that overexpress cMet, we inserted the HGF NK2 sequence, corresponding to a competitive antagonist of HGF binding to the cMet receptor, into the Ad serotype 5 (Ad5) fiber gene. The resulting vector, Ad5-pIX-RFP-FF/NK2, was rescued, amplified in HEK293 cells, and characterized. Binding specificity and viral infectivity were tested in various cancer cell lines that express varying levels of cMet and hCAR (the Ad5 receptor). We found that Ad5-pIX-RFP-FF/NK2 demonstrated binding specificity to the cMet receptor. In addition, there was enhanced viral infectivity and virus replication compared with a non-targeted Ad vector. Although NK2 weakly induces cMet receptor activation, our results showed no receptor phosphorylation in the context of an oncolytic Ad virus. In summary, these results suggest that an oncolytic Ad retargeted to the cMet receptor is a promising vector for developing a novel cancer therapeutic agent. PMID:26866014

  15. Gene transfer vectors targeted to human prostate cancer: do we need better preclinical testing systems?

    PubMed

    Maitland, Norman; Chambers, Karen; Georgopoulos, Lindsay; Simpson-Holley, Martha; Leadley, Regina; Evans, Helen; Essand, Magnus; Danielsson, Angelika; van Weerden, Wytske; de Ridder, Corrina; Kraaij, Robert; Bangma, Chris H

    2010-07-01

    Destruction of cancer cells by genetically modified viral and nonviral vectors has been the aim of many research programs. The ability to target cytotoxic gene therapies to the cells of interest is an essential prerequisite, and the treatment has always had the potential to provide better and more long-lasting therapy than existing chemotherapies. However, the potency of these infectious agents requires effective testing systems, in which hypotheses can be explored both in vitro and in vivo before the establishment of clinical trials in humans. The real prospect of off-target effects should be eliminated in the preclinical stage, if current prejudices against such therapies are to be overcome. In this review we have set out, using adenoviral vectors as a commonly used example, to discuss some of the key parameters required to develop more effective testing, and to critically assess the current cellular models for the development and testing of prostate cancer biotherapy. Only by developing models that more closely mirror human tissues will we be able to translate literature publications into clinical trials and hence into acceptable alternative treatments for the most commonly diagnosed cancer in humans.

  16. An Improved Systematic Approach to Predicting Transcription Factor Target Genes Using Support Vector Machine

    PubMed Central

    Cui, Song; Youn, Eunseog; Lee, Joohyun; Maas, Stephan J.

    2014-01-01

    Biological prediction of transcription factor binding sites and their corresponding transcription factor target genes (TFTGs) makes great contribution to understanding the gene regulatory networks. However, these approaches are based on laborious and time-consuming biological experiments. Numerous computational approaches have shown great potential to circumvent laborious biological methods. However, the majority of these algorithms provide limited performances and fail to consider the structural property of the datasets. We proposed a refined systematic computational approach for predicting TFTGs. Based on previous work done on identifying auxin response factor target genes from Arabidopsis thaliana co-expression data, we adopted a novel reverse-complementary distance-sensitive n-gram profile algorithm. This algorithm converts each upstream sub-sequence into a high-dimensional vector data point and transforms the prediction task into a classification problem using support vector machine-based classifier. Our approach showed significant improvement compared to other computational methods based on the area under curve value of the receiver operating characteristic curve using 10-fold cross validation. In addition, in the light of the highly skewed structure of the dataset, we also evaluated other metrics and their associated curves, such as precision-recall curves and cost curves, which provided highly satisfactory results. PMID:24743548

  17. Receptor-targeted recombinant adenovirus conglomerates: a novel molecular conjugate vector with improved expression characteristics.

    PubMed Central

    Schwarzenberger, P; Hunt, J D; Robert, E; Theodossiou, C; Kolls, J K

    1997-01-01

    To develop improved strategies for gene transfer to hematopoietic cells, we have explored targeted gene transfer using molecular conjugate vectors (MCVs). MCVs are constructed by condensing plasmid DNA containing the gene of interest with polylysine (PL), PL linked to a replication-incompetent adenovirus (endosomolytic agent), and PL linked to streptavidin for targeting with biotinylated ligands. In this report, we compare gene transfer to K562 cells by using the previously described transferrin-targeted MCV (Trans-MCV) to a novel transferrin-targeted MCV. In the novel MCV, the transferred gene (luciferase) is in the genome of recombinant replication-incompetent adenovirus (recMCV), which also acts as the endosomolytic agent. The level of luciferase gene expression was fivefold higher in K562 cells transfected with Trans-recMCV than in cells transfected with Trans-MCV. Furthermore, targeted transfection with recMCV resulted in prolonged luciferase expression that declined 14 to 20 days after transfection, in comparison with Trans-MCV, where luciferase expression declined by 4 to 8 days. Moreover, targeted transfection of K562 cells with the Trans-recMCV resulted in persistent luciferase gene expression for 6 months. Analysis of luciferase gene expression in K562 single-cell clones that were subcloned 5 weeks after transfection with Trans-recMCV showed that 35 to 50% of the single-cell clones had intermediate to high levels of luciferase gene expression that was stable for 6 months, with the remaining clones showing low or no luciferase gene expression. Stable gene expression was associated with integration of adenovirus sequences into genomic DNA. PMID:9343214

  18. Transcriptional targeting of primary and metastatic tumor neovasculature by an adenoviral type 5 roundabout4 vector in mice.

    PubMed

    Lu, Zhi Hong; Kaliberov, Sergey; Sohn, Rebecca E; Kaliberova, Lyudmila; Curiel, David T; Arbeit, Jeffrey M

    2013-01-01

    New approaches targeting metastatic neovasculature are needed. Payload capacity, cellular transduction efficiency, and first-pass cellular uptake following systemic vector administration, motivates persistent interest in tumor vascular endothelial cell (EC) adenoviral (Ad) vector targeting. While EC transductional and transcriptional targeting has been accomplished, vector administration approaches of limited clinical utility, lack of tumor-wide EC expression quantification, and failure to address avid liver sequestration, challenged prior work. Here, we intravenously injected an Ad vector containing 3 kb of the human roundabout4 (ROBO4) enhancer/promoter transcriptionally regulating an enhanced green fluorescent protein (EGFP) reporter into immunodeficient mice bearing 786-O renal cell carcinoma subcutaneous (SC) xenografts and kidney orthotopic (KO) tumors. Initial experiments performed in human coxsackie virus and adenovirus receptor (hCAR) transgenic:Rag2 knockout mice revealed multiple ECs with high-level Ad5ROBO4-EGFP expression throughout KO and SC tumors. In contrast, Ad5CMV-EGFP was sporadically expressed in a few tumor vascular ECs and stromal cells. As the hCAR transgene also facilitated Ad5ROBO4 and control Ad5CMV vector EC expression in multiple host organs, follow-on experiments engaged warfarin-mediated liver vector detargeting in hCAR non-transgenic mice. Ad5ROBO4-mediated EC expression was undetectable in most host organs, while the frequencies of vector expressing intratumoral vessels and whole tumor EGFP protein levels remained elevated. In contrast, AdCMV vector expression was only detectable in one or two stromal cells throughout the whole tumor. The Ad5ROBO4 vector, in conjunction with liver detargeting, provides tractable genetic access for in-vivo EC genetic engineering in malignancies.

  19. Transcriptional Targeting of Primary and Metastatic Tumor Neovasculature by an Adenoviral Type 5 Roundabout4 Vector in Mice

    PubMed Central

    Lu, Zhi Hong; Kaliberov, Sergey; Sohn, Rebecca E.; Kaliberova, Lyudmila; Curiel, David T.; Arbeit, Jeffrey M.

    2013-01-01

    New approaches targeting metastatic neovasculature are needed. Payload capacity, cellular transduction efficiency, and first-pass cellular uptake following systemic vector administration, motivates persistent interest in tumor vascular endothelial cell (EC) adenoviral (Ad) vector targeting. While EC transductional and transcriptional targeting has been accomplished, vector administration approaches of limited clinical utility, lack of tumor-wide EC expression quantification, and failure to address avid liver sequestration, challenged prior work. Here, we intravenously injected an Ad vector containing 3 kb of the human roundabout4 (ROBO4) enhancer/promoter transcriptionally regulating an enhanced green fluorescent protein (EGFP) reporter into immunodeficient mice bearing 786-O renal cell carcinoma subcutaneous (SC) xenografts and kidney orthotopic (KO) tumors. Initial experiments performed in human coxsackie virus and adenovirus receptor (hCAR) transgenic:Rag2 knockout mice revealed multiple ECs with high-level Ad5ROBO4-EGFP expression throughout KO and SC tumors. In contrast, Ad5CMV-EGFP was sporadically expressed in a few tumor vascular ECs and stromal cells. As the hCAR transgene also facilitated Ad5ROBO4 and control Ad5CMV vector EC expression in multiple host organs, follow-on experiments engaged warfarin-mediated liver vector detargeting in hCAR non-transgenic mice. Ad5ROBO4-mediated EC expression was undetectable in most host organs, while the frequencies of vector expressing intratumoral vessels and whole tumor EGFP protein levels remained elevated. In contrast, AdCMV vector expression was only detectable in one or two stromal cells throughout the whole tumor. The Ad5ROBO4 vector, in conjunction with liver detargeting, provides tractable genetic access for in-vivo EC genetic engineering in malignancies. PMID:24376772

  20. Radar target classification method with high accuracy and decision speed performance using MUSIC spectrum vectors and PCA projection

    NASA Astrophysics Data System (ADS)

    Secmen, Mustafa

    2011-10-01

    This paper introduces the performance of an electromagnetic target recognition method in resonance scattering region, which includes pseudo spectrum Multiple Signal Classification (MUSIC) algorithm and principal component analysis (PCA) technique. The aim of this method is to classify an "unknown" target as one of the "known" targets in an aspect-independent manner. The suggested method initially collects the late-time portion of noise-free time-scattered signals obtained from different reference aspect angles of known targets. Afterward, these signals are used to obtain MUSIC spectrums in real frequency domain having super-resolution ability and noise resistant feature. In the final step, PCA technique is applied to these spectrums in order to reduce dimensionality and obtain only one feature vector per known target. In the decision stage, noise-free or noisy scattered signal of an unknown (test) target from an unknown aspect angle is initially obtained. Subsequently, MUSIC algorithm is processed for this test signal and resulting test vector is compared with feature vectors of known targets one by one. Finally, the highest correlation gives the type of test target. The method is applied to wire models of airplane targets, and it is shown that it can tolerate considerable noise levels although it has a few different reference aspect angles. Besides, the runtime of the method for a test target is sufficiently low, which makes the method suitable for real-time applications.

  1. Optimization and internalization mechanisms of PEGylated adenovirus vector with targeting peptide for cancer gene therapy.

    PubMed

    Yao, Xing-Lei; Yoshioka, Yasuo; Ruan, Gui-Xin; Chen, Yu-Zhe; Mizuguchi, Hiroyuki; Mukai, Yohei; Okada, Naoki; Gao, Jian-Qing; Nakagawa, Shinsaku

    2012-08-13

    We have previously developed a novel adenovirus vector (Adv) that targeted tumor tissues/vasculatures after systemic administration. The surface of this Adv is conjugated with CGKRK tumor homing peptide by the cross-linking reaction of polyethyleneglycol (PEG). In this study, we showed that the condition of PEG modification was important to minimize the gene expression in normal tissues after systemic treatment. When Adv was modified only with PEG-linked CGKRK, its luciferase expression was enhanced even in the liver tissue, as well as the tumor tissue. However, in the reaction with the mixture of non-cross-linking PEG and PEG-linked CGKRK, we found out that the best modification could suppress its gene expression in the liver, without losing that in the tumor. We also studied the internalization mechanisms of CGKRK-conjugated Adv. Results suggested that there is a specific interaction of the CGKRK peptide with a receptor at the cell surface enabling efficient internalization of CGKRK-conjugated Adv. The presence of cell-surface heparan sulfate is important receptor for the cellular binding and uptake of CGKRK-conjugated Adv. Moreover, macropinocytosis-mediated endocytosis is also important in endocytosis of CGKRK-conjugated Adv, aside from clathrin-mediated and caveolae-mediated endocytosis. These results could help evaluate the potentiality of CGKRK-conjugated Adv as a prototype vector with suitable efficacy and safety for systemic cancer gene therapy.

  2. Preparation and characterization of magnetic gene vectors for targeting gene delivery

    NASA Astrophysics Data System (ADS)

    Zheng, S. W.; Liu, G.; Hong, R. Y.; Li, H. Z.; Li, Y. G.; Wei, D. G.

    2012-10-01

    The PEI-CMD-MNPs were successfully prepared by the surface modification of magnetic Fe3O4 nanoparticles with carboxymethyl dextran (CMD) and polyethyleneimine (PEI). The PEI-CMD-MNPs polyplexes exhibited a typical superparamagnetic behavior and were well stable over the entire range of pH and NaCl concentration. These PEI-CMD-MNPs were used as magnetic gene vectors for targeting gene delivery. The prepared MNPs at different surface modification stages were characterized using Fourier transform infrared (FT-IR), thermogravimetric analysis (TGA), field emissions canning electron microscopy (FE-SEM), powder X-ray diffraction (XRD) and dynamic laser light scattering (DLS) analysis. The magnetic properties were studied by vibrating sample magnetometer (VSM). To evaluate the performance of the magnetic nanoparticles as gene transfer vector, the PEI-CMD-MNPs were used to delivery green fluorescent protein (GFP) gene into BHK21 cells. The expression of GFP gene was detected by fluorescence microscope. DNA-PEI-CMD-MNPs polyplexes absorbed by the cells were also monitored by Magnetic resonance imaging (MRI). The transfection efficiency and gene expression efficiency of that transfected with a magnet were much higher than that of standard transfection.

  3. Targeted expression of two proteins in neural retina using self-inactivating, insulated lentiviral vectors carrying two internal independent promoters.

    PubMed

    Semple-Rowland, Susan L; Eccles, Kristofer S; Humberstone, Elizabeth J

    2007-10-18

    There is increasing interest in developing viral vectors capable of reliably delivering multiple therapeutic genes to targeted cell populations. Currently, bicistronic vectors carrying two transgenes linked by an internal ribosomal entry site (IRES) are the most commonly employed vectors to accomplish this goal. We and others have found that the protein encoded downstream of the IRES in these vectors is not reliably expressed. The purpose of this study was to determine if replacement of the IRES in our self-inactivating, insulated, lentiviral vectors with a second, independent, cell-specific promoter would produce a vector that reliably expressed two proteins in targeted retinal cells in vivo. Five dual promoter lentiviral vectors were constructed using our self-inactivating (SIN), insulated, lentiviral backbone. Each vector carried two independent transgenes encoding a fluorescent protein (GFP or tdTomato) whose expression was driven by three photoreceptor promoters (interphotoreceptor retinoid binding protein-IRPB1783; guanylate cyclase activating protein 1-GCAP292; rhodopsin-mOP500) and one ubiquitously expressed promoter (elongation factor 1alpha-EF1alpha). Constructs were packaged and injected into the optic vesicles of developing chicken embryos. The day before hatching, the retinas were removed and examined as whole mount tissues and as frozen sections using fluorescent microscopy. In our first experiment, we characterized the expression of the three photoreceptor promoters in chicken retina. The activities of GCAP292 and IRBP1783 were restricted to cone cells. GCAP292 was also active in a small sub-group of inner nuclear cells. The activity of mOP500 was restricted to rod cells. In our second experiment, we characterized the activity of three dual promoter vectors: GCAP292-GFP-IRBP1783-tdTomato, IRBP-tdTomato-GCAP292-GFP, and IRBP1783-tdTomato-mOP500-GFP. All three vectors produced easily detectable levels of GFP and tdTomato in transduced retinas, a result

  4. Theranostic Value of Multimers: Lessons Learned from Trimerization of Neurotensin Receptor Ligands and Other Targeting Vectors

    PubMed Central

    Maschauer, Simone; Einsiedel, Jürgen; Reich, Dominik; Hübner, Harald; Gmeiner, Peter; Wester, Hans-Jürgen; Prante, Olaf; Notni, Johannes

    2017-01-01

    Neurotensin receptor 1 (NTS1) is overexpressed on a variety of cancer entities; for example, prostate cancer, ductal pancreatic adenocarcinoma, and breast cancer. Therefore, it represents an interesting target for the diagnosis of these cancers types by positron emission tomography (PET). The metabolically-stabilized neurotensin (NT) derivative peptide Nlys8-Lys9-Pro10-Tyr11-Tle12-Leu13-OH was elongated at the N-terminus with 6-azido norleucine and coupled with the 1,4,7-triazacyclononane-1,4,7-tris[(2-carboxyethyl)methylenephosphinic acid] (TRAP) chelator TRAP(alkyne)3 in order to synthesize a NT trimer with subnanomolar affinity and high stability. The 68Ga-labeled peptide [68Ga]Ga-TRAP(NT4)3 was characterized in vitro using the NTS1-expressing human colorectal adenocarcinoma cell line HT29. It displayed fast and high internalization rates of >90%, but also fast efflux rates of 50% over 15 min. In vivo, [68Ga]Ga-TRAP(NT4)3 showed moderate HT29 tumor uptake values of 1.7 %ID/g at 60 min post-injection (p.i.), but also high uptake and retention in the kidneys and liver. A comparison of data for trimer/monomer pairs of NT ligands and other targeting vectors (peptides and peptoids targeting integrins αvβ3, α5β1, and αvβ6, the PSMA-ligand DUPA (2-[3-(1,3-dicarboxypropyl)-ureido]pentanedioic acid), and nitroimidazoles targeting hypoxia) revealed that multimers always exhibit higher target affinities and tumor uptake, but not necessarily improved tumor-to-tissue ratios. Thus, although in vitro data are not suitable for prediction of in vivo performance, multimers are potentially superior to monomers, particularly for applications where high tumor accumulation is crucial. PMID:28287433

  5. Surface engineering of lentiviral vectors for gene transfer into gene therapy target cells.

    PubMed

    Lévy, Camille; Verhoeyen, Els; Cosset, François-Loïc

    2015-10-01

    Since they allow gene integration into their host genome, lentiviral vectors (LVs) have strong therapeutic potentials, as emphasized by recent clinical trials. The surface-display of the pantropic vesicular stomatitis virus G glycoprotein (VSV-G) on LVs resulted in powerful tools for fundamental and clinical research. However, improved LVs are required either to genetically modify cell types not permissive to classical VSV-G-LVs or to restrict entry to specific cell types. Incorporation of heterologous viral glycoproteins (gps) on LVs often require modification of their cytoplasmic tails and ligands can be inserted into their ectodomain to target LVs to specific receptors. Recently, measles virus (MV) gps have been identified as strong candidates for LV-retargeting to multiple cell types, with the potential to evolve toward clinical applications. Copyright © 2015 Elsevier Ltd. All rights reserved.

  6. Cytomegalovirus and immunotherapy: opportunistic pathogen, novel target for cancer and a promising vaccine vector.

    PubMed

    Quinn, Michael; Erkes, Dan A; Snyder, Christopher M

    2016-02-01

    Cytomegalovirus (CMV) is a β-herpesvirus that infects most people in the world and is almost always asymptomatic in the healthy host. However, CMV persists for life, requiring continuous immune surveillance to prevent disease and thus, CMV is a frequent complication in immune compromised patients. Many groups have been exploring the potential for adoptive T-cell therapies to control CMV reactivation as well as the progression of solid tumors harboring CMV. In addition, CMV itself is being explored as a vaccine vector for eliciting potent T-cell responses. This review will discuss key features of the basic biology of CMV-specific T cells as well as highlighting unanswered questions and ongoing work in the development of T-cell-based immunotherapies to target CMV.

  7. A Vector-Based Short Hairpin RNA Targeting Aurora B Suppresses Human Prostatic Carcinoma Growth.

    PubMed

    Cao, Mei; Qi, Panpan; Chen, Chong; Song, Liju; Wang, Xuege; Li, Ningzhe; Wu, Daoyan; Hu, Guoku; Zhao, Jian

    2017-02-01

    Aurora kinase B, playing a vital, important role in mitosis, is frequently detected to be overexpressed in many cancer cell lines and various tumor tissues, including prostatic carcinoma. Given the essential function of Aurora kinase B in mitosis and its association with tumorigenesis, it might be a drug target for prostatic carcinoma treatment. In our study, short hairpin RNA targeting Aurora kinase B was cloned into a pGPU6 plasmid vector and then transfected into human prostatic carcinoma cells. The expression level of Aurora kinase B was verified by reverse transcription-polymerase chain reaction and Western blot. At the same time, cell apoptosis was detected by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide, fluorescent staining, and flow cytometric analysis. Furthermore, prostate carcinoma cells were injected into mice to establish a tumor xenograft model. Previous studies have shown the effect of pGPU6-shAURKB plasmid on tumor growth in a prostate carcinoma xenogenic implantation model. From the study, we knew that the Aurora kinase B was significantly downregulated in prostate carcinoma cells, and cell apoptosis was also detected higher in treated groups than that in control groups. Moreover, in the prostate carcinoma xenogenic implantation model, compared with the control groups, the tumor growth was inhibited about 78.7% in the pGPU6-shAURKB plasmid-treated group, and cell apoptosis in the experimental group was notably higher than that in control groups. The average duration of tumor-bearing mice was prolonged to about 35 days. The results of experiment indicated that specific knockdown of Aurora kinase B led to prostate carcinoma cells apoptosis and inhibited tumor growth. Our data clearly confirmed that specific knockdown of Aurora kinase B expression by vector-based short hairpin RNA/liposome may be a potential new approach to treat human prostatic carcinoma.

  8. A new method for rapidly generating gene-targeting vectors by engineering BACs through homologous recombination in bacteria.

    PubMed

    Cotta-de-Almeida, Vinicius; Schonhoff, Susan; Shibata, Tomoyuki; Leiter, Andrew; Snapper, Scott B

    2003-09-01

    Generating knockout mice is still an expensive and highly time-consuming process. Target construct generation, the first labor-intensive step in this process, requires the manipulation of large fragments of DNA and numerous, and often cumbersome, cloning steps. Here we show the development of a rapid approach for generating targeting constructs that capitalizes on efficient homologous recombination between linear DNA fragments and circular plasmids in Escherichia coli ("recombineering"), the availability of bacterial artificial chromosomes (BACs), and the accessibility of the sequence of the mouse genome. Employing recombineering, we demonstrate with only 1-2 template plasmids, short homologies (40-50bp) between donor and target DNA, and one subcloning step that we can efficiently manipulate BACs in situ to generate a complicated targeting vector. This procedure avoids the need to construct or screen genomic libraries and permits the generation of most standard, conditional, or knock-in targeting vectors, often within two weeks.

  9. Using Support Vector Machine Ensembles for Target Audience Classification on Twitter

    PubMed Central

    Lo, Siaw Ling; Chiong, Raymond; Cornforth, David

    2015-01-01

    The vast amount and diversity of the content shared on social media can pose a challenge for any business wanting to use it to identify potential customers. In this paper, our aim is to investigate the use of both unsupervised and supervised learning methods for target audience classification on Twitter with minimal annotation efforts. Topic domains were automatically discovered from contents shared by followers of an account owner using Twitter Latent Dirichlet Allocation (LDA). A Support Vector Machine (SVM) ensemble was then trained using contents from different account owners of the various topic domains identified by Twitter LDA. Experimental results show that the methods presented are able to successfully identify a target audience with high accuracy. In addition, we show that using a statistical inference approach such as bootstrapping in over-sampling, instead of using random sampling, to construct training datasets can achieve a better classifier in an SVM ensemble. We conclude that such an ensemble system can take advantage of data diversity, which enables real-world applications for differentiating prospective customers from the general audience, leading to business advantage in the crowded social media space. PMID:25874768

  10. Library selection and directed evolution approaches to engineering targeted viral vectors.

    PubMed

    Jang, Jae-Hyung; Lim, Kwang-il; Schaffer, David V

    2007-10-15

    Gene therapy, to delivery of genetic material to a patient for therapeutic benefit, has significant promise for translating basic knowledge of disease mechanism into biomedical treatments. The clinical development of the field has been slowed, however, by the need for improvements in the properties and capabilities of gene delivery vehicles. Vehicles based on viruses offer the potential for efficient gene delivery, but because viruses did not evolve to serve human therapeutic needs, many of their properties require significant improvement, including their safety, efficiency, and capacity for targeted gene delivery. Since viruses are highly complex biological entities, engineering such properties at the molecular level can be challenging. However, there has been significant progress in developing approaches that mimic the mechanisms by which viruses arose in the first place. In particular, library-based selection, the generation of one diverse genetic library and selection for new properties, and directed evolution, based on the multiple rounds of library generation and selection for iterative improvement of function, have strong potential in engineering novel properties into these complex biomolecular assemblies. This review will discuss progress in the application of peptide display, library selection, and directed evolution technologies toward engineering vectors based on retrovirus, adeno-associated virus, and adenovirus that are capable of targeted delivery to specific cell types. In addition to creating biomedically useful products, these approaches have future potential to yield novel insights into viral structure-function relationships. Copyright 2007 Wiley Periodicals, Inc.

  11. Using support vector machine ensembles for target audience classification on Twitter.

    PubMed

    Lo, Siaw Ling; Chiong, Raymond; Cornforth, David

    2015-01-01

    The vast amount and diversity of the content shared on social media can pose a challenge for any business wanting to use it to identify potential customers. In this paper, our aim is to investigate the use of both unsupervised and supervised learning methods for target audience classification on Twitter with minimal annotation efforts. Topic domains were automatically discovered from contents shared by followers of an account owner using Twitter Latent Dirichlet Allocation (LDA). A Support Vector Machine (SVM) ensemble was then trained using contents from different account owners of the various topic domains identified by Twitter LDA. Experimental results show that the methods presented are able to successfully identify a target audience with high accuracy. In addition, we show that using a statistical inference approach such as bootstrapping in over-sampling, instead of using random sampling, to construct training datasets can achieve a better classifier in an SVM ensemble. We conclude that such an ensemble system can take advantage of data diversity, which enables real-world applications for differentiating prospective customers from the general audience, leading to business advantage in the crowded social media space.

  12. Helper-dependent adenoviral vectors are superior in vitro to first-generation vectors for endothelial cell-targeted gene therapy.

    PubMed

    Flynn, Rowan; Buckler, Joshua M; Tang, Chongren; Kim, Francis; Dichek, David A

    2010-12-01

    Arterial endothelial cells (EC) are attractive targets for gene therapy of atherosclerosis because they are accessible to hematogenous and catheter-based vector delivery and overlie atherosclerotic plaques. Vector-mediated expression-in EC-of proteins that mediate cholesterol transfer out of the artery wall and decrease inflammation could prevent and reverse atherosclerosis. However, clinical application of this strategy is limited by lack of a suitable gene-transfer vector. First-generation adenovirus (FGAd) is useful for EC gene transfer in proof-of-concept studies, but is unsuitable for atheroprotective human gene therapy because of limited duration of expression and proinflammatory effects. Moreover, others have reported detrimental effects of FGAd on critical aspects of EC physiology including proliferation, migration, and apoptosis. Here, we investigated whether helper-dependent adenovirus (HDAd) either alone or expressing an atheroprotective gene [apolipoprotein A-I (apoA-I)] could circumvent these limitations. In contrast to control FGAd, HDAd did not alter any of several critical EC physiologic functions (including proliferation, migration, apoptosis, metabolic activity, and nitric oxide (NO) production) and did not stimulate proinflammatory pathways [including expression of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and interleukin-6 (IL-6)]. Expression of apoA-I by HDAd reduced EC VCAM-1 expression. HDAd is a promising vector and apoA-I is a promising gene for atheroprotective human gene therapy delivered via EC.

  13. Regulation of the Target Protein (Transgene) Expression in the Adenovirus Vector Using Agonists of Toll-Like Receptors

    PubMed Central

    Bagaev, A. V.; Pichugin, A. V.; Lebedeva, E. S.; Lysenko, A. A.; Shmarov, M. M.; Logunov, D. Yu.; Naroditsky, B. S.; Ataullakhanov, R. I.; Khaitov, R. M.; Gintsburg, A. L.

    2014-01-01

    Replication-defective adenoviral vectors are effective molecular tools for both gene therapy and gene vaccination. Using such vectors one can deliver and express target genes in different epithelial, liver, hematopoietic and immune system cells of animal and human origin. The success of gene therapy and gene vaccination depends on the production intensity of the target protein encoded by the transgene. In this work, we studied influence of Toll-like receptors (TLR) agonists on transduction and expression efficacy of adenoviral vectors in animal and human antigen-presenting cells. We found that agonists of TLR2, 4, 5, 7, 8 and 9 significantly enhance a production of the target protein in cells transduced with adenoviral vector having the target gene insert. The enhancement was observed in dendritic cells and macrophages expressing cytoplasmic (GFP), membrane (HA) or secretory (SEAP) proteins encoded by the respective rAd-vectors. Experiments in mice showed that enhancement of the transgene expression can be achieved in the organism of animals using a pharmaceutical-grade TLR4-agonist. In contrast to other TLR-agonists, the agonist of TLR3 substantially suppressed the expression of transgene in cells transduced with adenoviral vectors having insert of GFP or SEAP target genes. We propose that the enhancement of transgene expression is linked to the activation of MyD88→ NF-kB, while the inhibition of transgene expression depends on TRIF→ IRF signaling pathways. Both of these pathways jointly exploited by TLR4-agonists lead to the enhancement of transgene expression due to the dominant role of the MyD88→ NF-kB signaling. PMID:25558392

  14. Novel hyaluronic acid-chitosan nanoparticles as non-viral gene delivery vectors targeting osteoarthritis.

    PubMed

    Lu, Hua-Ding; Zhao, Hui-Qing; Wang, Kun; Lv, Lu-Lu

    2011-11-28

    Gene therapy is a promising new treatment strategy for common joint-disorders such as osteoarthritis. The development of safe, effective, targeted non-viral gene carriers is important for the clinical success of gene therapy. The present work describes the use of hybrid hyaluronic acid (HA)/chitosan (CS) nanoparticles as novel non-viral gene delivery vectors capable of transferring exogenous genes into primary chondrocytes for the treatment of joint diseases. HA/CS plasmid-DNA nanoparticles were synthesized through the complex coacervation of the cationic polymers with pEGFP. Particle size and zeta potential were related to the weight ratio of CS to HA, where increases in nanoparticle size and decreases in surface charge were observed as HA content increased. The particle size and the zeta potential varied according to pH. Transfection of primary chondrocytes was performed under different conditions to examine variations in the pH of the transfection medium, different N/P ratios, different plasmid concentrations, and different molecular weights of chitosan. Transfection efficiency was maximized for a medium pH of approximately 6.8, an N/P ratio of 5, plasmid concentration of 4 μg/ml, and a chitosan molecular weight of 50 kDa. The transfection efficiency of HA/CS-plasmid nanoparticles was significantly higher than that of CS-plasmid nanoparticles under the same conditions. The average viability of cells transfected with HA/CS-plasmid nanoparticles was over 90%. These results suggest that HA/CS-plasmid nanoparticles could be an effective non-viral vector suitable for gene delivery to chondrocytes.

  15. Liver-specific microRNA-122 target sequences incorporated in AAV vectors efficiently inhibits transgene expression in the liver.

    PubMed

    Qiao, C; Yuan, Z; Li, J; He, B; Zheng, H; Mayer, C; Li, J; Xiao, X

    2011-04-01

    Vectors based on adeno-associated virus (AAV) are effective in gene delivery in vivo. Tissue-specific gene expression is often needed to minimize ectopic expression in unintended cells and undesirable consequences. Here, we investigated whether incorporation of target sequences of tissue-specific microRNA (miRNA) into AAV vectors could inhibit ectopic expression in tissues such as the liver and hematopoietic cells. First we inserted liver-specific miR-122 target sequences (miR-122T) into the 3'-untranslated region (UTR) of a number of AAV vectors. After intravenous delivery in mice, we found that five copies of the 20mer miR-122T reduced liver expression of luciferase by 50-fold and β-galactosidase (LacZ) by 70-fold. Five copies of miR-122T also reduced mRNA levels of a secretable protein (myostatin propeptide) from the AAV vector plasmid by 23-fold in the liver. However, gene expression in other tissues, including the heart was not inhibited. Similarly, we inserted four copies of miR-142-3pT or miR-142-5pT, both hematopoietic lineage-specific, into the 3'-UTR of the AAV-luciferase vector. We wished to see whether they could prolong transgene expression by inhibiting expression in antigen-presenting cells. However, in vivo luciferase gene expression in major tissues declined with time, regardless of the miR-142 target sequences used. Quantitative analysis of the vector DNA in various tissues revealed that the decline of transgene expression in vivo was mainly because of promoter shut-off other than loss of AAV-transduced cells by immune destruction. Moreover, transgene expression was not detected in circulating mononuclear cells after delivering AAV9 vector with or without miR142T. These results demonstrate that liver-specific miR-122 target sequence in AAV vectors was highly efficient in reducing liver expression, whereas hematopoietic miR-142 target sequences were ineffective in preventing decline of AAV vector gene expression in nonhematopoietic tissues

  16. Clustered Integrin Ligands as a Novel Approach for the Targeting of Non-Viral Vectors

    NASA Astrophysics Data System (ADS)

    Ng, Quinn Kwan Tai

    Gene transfer or gene delivery is described as the process in which foreign DNA is introduced into cells. Over the years, gene delivery has gained the attention of many researchers and has been developed as powerful tools for use in biotechnology and medicine. With the completion of the Human Genome Project, such advances in technology allowed for the identification of diseases ranging from hereditary disorders to acquired ones (cancer) which were thought to be incurable. Gene therapy provides the means necessary to treat or eliminate genetic diseases from its origin, unlike traditional medicine which only treat symptoms. With ongoing clinical trials for gene therapy increasing, the greatest difficulty still lies in developing safe systems which can target cells of interest to provide efficient delivery. Nature, over millions of years of evolution, has provided an example of one of the most efficient delivery systems: viruses. Although the use of viruses for gene delivery has been well studied, the safety issues involving immunogenicity, insertional mutagenesis, high cost, and poor reproducibility has provided problems for their clinical application. From understanding viruses, we gain insight to designing new systems for non-viral gene delivery. One of these techniques utilized by adenoviruses is the clustering of ligands on its surface through the use of a protein called a penton base. Through the use of nanotechnology we can mimic this basic concept in non-viral gene delivery systems. This dissertation research is focused on developing and applying a novel system for displaying the integrin binding ligand (RGD) in a constrained manner to form a clustered integrin ligand binding platform to be used to enhance the targeting and efficiency of non-viral gene delivery vectors. Peptide mixed monolayer protected gold nanoparticles provides a suitable surface for ligand clustering. A relationship between the peptide ratios in the reaction solution used to form these

  17. Optimization and in Vivo Validation of Peptide Vectors Targeting the LDL Receptor.

    PubMed

    Jacquot, Guillaume; Lécorché, Pascaline; Malcor, Jean-Daniel; Laurencin, Mathieu; Smirnova, Maria; Varini, Karine; Malicet, Cédric; Gassiot, Fanny; Abouzid, Karima; Faucon, Aude; David, Marion; Gaudin, Nicolas; Masse, Maxime; Ferracci, Géraldine; Dive, Vincent; Cisternino, Salvatore; Khrestchatisky, Michel

    2016-12-05

    in wild-type or ldlr -/- mice confirmed their active LDLR targeting in vivo. Overall, this study extends our previous work toward a diversified portfolio of LDLR-targeted peptide vectors with validated LDLR-targeting potential in vivo.

  18. Target Localization in Wireless Sensor Networks Using Online Semi-Supervised Support Vector Regression

    PubMed Central

    Yoo, Jaehyun; Kim, H. Jin

    2015-01-01

    Machine learning has been successfully used for target localization in wireless sensor networks (WSNs) due to its accurate and robust estimation against highly nonlinear and noisy sensor measurement. For efficient and adaptive learning, this paper introduces online semi-supervised support vector regression (OSS-SVR). The first advantage of the proposed algorithm is that, based on semi-supervised learning framework, it can reduce the requirement on the amount of the labeled training data, maintaining accurate estimation. Second, with an extension to online learning, the proposed OSS-SVR automatically tracks changes of the system to be learned, such as varied noise characteristics. We compare the proposed algorithm with semi-supervised manifold learning, an online Gaussian process and online semi-supervised colocalization. The algorithms are evaluated for estimating the unknown location of a mobile robot in a WSN. The experimental results show that the proposed algorithm is more accurate under the smaller amount of labeled training data and is robust to varying noise. Moreover, the suggested algorithm performs fast computation, maintaining the best localization performance in comparison with the other methods. PMID:26024420

  19. Evaluation of CD46 re-targeted adenoviral vectors for clinical ovarian cancer intraperitoneal therapy

    PubMed Central

    Hulin-Curtis, S L; Uusi-Kerttula, H; Jones, R; Hanna, L; Chester, J D; Parker, A L

    2016-01-01

    Ovarian cancer accounts for >140 000 deaths globally each year. Typically, disease is asymptomatic until an advanced, incurable stage. Although response to cytotoxic chemotherapy is frequently observed, resistance to conventional platinum-based therapies develop rapidly. Improved treatments are therefore urgently required. Virotherapy offers great potential for ovarian cancer, where the application of local, intraperitoneal delivery circumvents some of the limitations of intravenous strategies. To develop effective, adenovirus (Ad)-based platforms for ovarian cancer, we profiled the fluid and cellular components of patient ascites for factors known to influence adenoviral transduction. Levels of factor X (FX) and neutralizing antibodies (nAbs) in ascitic fluid were quantified and tumor cells were assessed for the expression of coxsackie virus and adenovirus receptor (CAR) and CD46. We show that clinical ascites contains significant levels of FX but consistently high CD46 expression. We therefore evaluated in vitro the relative transduction of epithelial ovarian cancers (EOCs) by Ad5 (via CAR) and Ad5 pseudotyped with the fiber of Ad35 (Ad5T*F35++) via CD46. Ad5T*F35++ achieved significantly increased transduction in comparison to Ad5 (P<0.001), independent of FX and nAb levels. We therefore propose selective transduction of CD46 over-expressing EOCs using re-targeted, Ad35-pseudotyped Ad vectors may represent a promising virotherapy for ovarian cancer. PMID:27229159

  20. Tumor Vascular Targeted Delivery of Polymer-conjugated Adenovirus Vector for Cancer Gene Therapy

    PubMed Central

    Yao, Xinglei; Yoshioka, Yasuo; Morishige, Tomohiro; Eto, Yusuke; Narimatsu, Shogo; Kawai, Yasuaki; Mizuguchi, Hiroyuki; Gao, Jian-Qing; Mukai, Yohei; Okada, Naoki; Nakagawa, Shinsaku

    2011-01-01

    Previously, we generated a cancer-specific gene therapy system using adenovirus vectors (Adv) conjugated to polyethylene glycol (Adv-PEG). Here, we developed a novel Adv that targets both tumor tissues and tumor vasculatures after systemic administration by conjugating CGKRK tumor vasculature homing peptide to the end of a 20-kDa PEG chain (Adv-PEGCGKRK). In a primary tumor model, systemic administration of Adv-PEGCGKRK resulted in ~500- and 100-fold higher transgene expression in tumor than that of unmodified Adv and Adv-PEG, respectively. In contrast, the transgene expression of Adv-PEGCGKRK in liver was about 400-fold lower than that of unmodified Adv, and was almost the same as that of Adv-PEG. We also demonstrated that transgene expression with Adv-PEGCGKRK was enhanced in tumor vessels. Systemic administration of Adv-PEGCGKRK expressing the herpes simplex virus thymidine kinase (HSVtk) gene (Adv-PEGCGKRK-HSVtk) showed superior antitumor effects against primary tumors and metastases with negligible side effects by both direct cytotoxic effects and inhibition of tumor angiogenesis. These results indicate that Adv-PEGCGKRK has potential as a prototype Adv with suitable efficacy and safety for systemic cancer gene therapy against both primary tumors and metastases. PMID:21673661

  1. Targeting Sindbis virus-based vectors to Fc receptor-positive cell types

    SciTech Connect

    Klimstra, William B.; Williams, Jacqueline C.; Ryman, Kate D.; Heidner, Hans W. . E-mail: hans.heidner@utsa.edu

    2005-07-20

    Some viruses display enhanced infection for Fc receptor (FcR)-positive cell types when complexed with virus-specific immunoglobulin (Ig). This process has been termed antibody-dependent enhancement of viral infection (ADE). We reasoned that the mechanism of ADE could be exploited and adapted to target alphavirus-based vectors to FcR-positive cell types. Towards this goal, recombinant Sindbis viruses were constructed that express 1 to 4 immunoglobulin-binding domains of protein L (PpL) as N-terminal extensions of the E2 glycoprotein. PpL is a bacterial protein that binds the variable region of antibody kappa light chains from a range of mammalian species. The recombinant viruses incorporated PpL/E2 fusion proteins into the virion structure and recapitulated the species-specific Ig-binding phenotypes of native PpL. Virions reacted with non-immune serum or purified IgG displayed enhanced binding and ADE for several species-matched FcR-positive murine and human cell lines. ADE required virus expression of a functional PpL Ig-binding domain, and appeared to be Fc{gamma}R-mediated. Specifically, ADE did not occur with Fc{gamma}R-negative cells, did not require active complement proteins, and did not occur on Fc{gamma}R-positive murine cell lines when virions were bound by murine IgG-derived F(ab'){sub 2} fragments.

  2. Selective In Vivo Targeting of Human Liver Tumors by Optimized AAV3 Vectors in a Murine Xenograft Model

    PubMed Central

    Wang, Yuan; Zhang, Yuanhui; Ejjigani, Anila; Yin, Zifei; Lu, Yuan; Wang, Lina; Wang, Meng; Li, Jun; Hu, Zhongbo; Aslanidi, George V.; Zhong, Li; Gao, Guangping

    2014-01-01

    Abstract Current challenges for recombinant adeno-associated virus (rAAV) vector–based cancer treatment include the low efficiency and the lack of specificity in vivo. rAAV serotype 3 (rAAV3) vectors have previously been shown to be ineffective in normal mouse tissues following systemic administration. In the present study, we report that rAAV3 vectors can efficiently target and transduce various human liver cancer cells in vivo. Elimination of specific surface-exposed serine and threonine residues on rAAV3 capsids results in further augmentation in the transduction efficiency of these vectors, without any change in the viral tropism and cellular receptor interactions. In addition, we have identified a potential chemotherapy drug, shikonin, as a multifunctional compound to inhibit liver tumor growth as well as to significantly enhance the efficacy of rAAV vector-based gene therapy in vivo. Furthermore, we also document that suppression of tumorigenesis in a human liver cancer xenograft model can be achieved through systemic administration of the optimized rAAV3 vectors carrying a therapeutic gene, and shikonin at a dose that does not lead to liver damage. Our research provides a novel means to achieve not only targeted delivery but also the potential for gene therapy of human liver cancer. PMID:25296041

  3. BTK gene targeting by homologous recombination using a helper-dependent adenovirus/adeno-associated virus hybrid vector.

    PubMed

    Yamamoto, H; Ishimura, M; Ochiai, M; Takada, H; Kusuhara, K; Nakatsu, Y; Tsuzuki, T; Mitani, K; Hara, T

    2016-02-01

    X-linked agammaglobulinemia (XLA) is one of the most common humoral immunodeficiencies, which is caused by mutations in Bruton's tyrosine kinase (BTK) gene. To examine the possibility of using gene therapy for XLA, we constructed a helper-dependent adenovirus/adeno-associated virus BTK targeting vector (HD-Ad.AAV BTK vector) composed of a genomic sequence containing BTK exons 6-19 and a green fluorescence protein-hygromycin cassette driven by a cytomegalovirus promoter. We first used NALM-6, a human male pre-B acute lymphoblastic leukemia cell line, as a recipient to measure the efficiency of gene targeting by homologous recombination. We identified 10 clones with the homologous recombination of the BTK gene among 107 hygromycin-resistant stable clones isolated from two independent experiments. We next used cord blood CD34⁺ cells as the recipient cells for the gene targeting. We isolated colonies grown in medium containing cytokines and hygromycin. We found that the targeting of the BTK gene occurred in four of the 755 hygromycin-resistant colonies. Importantly, the gene targeting was also observed in CD19⁺ lymphoid progenitor cells that were differentiated from the homologous recombinant CD34⁺ cells during growth in selection media. Our study shows the potential for the BTK gene therapy using the HD-Ad.AAV BTK vector via homologous recombination in hematopoietic stem cells.

  4. Cre/lox-Recombinase-Mediated Cassette Exchange for Reversible Site-Specific Genomic Targeting of the Disease Vector, Aedes aegypti.

    PubMed

    Häcker, Irina; Harrell Ii, Robert A; Eichner, Gerrit; Pilitt, Kristina L; O'Brochta, David A; Handler, Alfred M; Schetelig, Marc F

    2017-03-07

    Site-specific genome modification (SSM) is an important tool for mosquito functional genomics and comparative gene expression studies, which contribute to a better understanding of mosquito biology and are thus a key to finding new strategies to eliminate vector-borne diseases. Moreover, it allows for the creation of advanced transgenic strains for vector control programs. SSM circumvents the drawbacks of transposon-mediated transgenesis, where random transgene integration into the host genome results in insertional mutagenesis and variable position effects. We applied the Cre/lox recombinase-mediated cassette exchange (RMCE) system to Aedes aegypti, the vector of dengue, chikungunya, and Zika viruses. In this context we created four target site lines for RMCE and evaluated their fitness costs. Cre-RMCE is functional in a two-step mechanism and with good efficiency in Ae. aegypti. The advantages of Cre-RMCE over existing site-specific modification systems for Ae. aegypti, phiC31-RMCE and CRISPR, originate in the preservation of the recombination sites, which 1) allows successive modifications and rapid expansion or adaptation of existing systems by repeated targeting of the same site; and 2) provides reversibility, thus allowing the excision of undesired sequences. Thereby, Cre-RMCE complements existing genomic modification tools, adding flexibility and versatility to vector genome targeting.

  5. Cre/lox-Recombinase-Mediated Cassette Exchange for Reversible Site-Specific Genomic Targeting of the Disease Vector, Aedes aegypti

    PubMed Central

    Häcker, Irina; Harrell II, Robert A.; Eichner, Gerrit; Pilitt, Kristina L.; O’Brochta, David A.; Handler, Alfred M.; Schetelig, Marc F.

    2017-01-01

    Site-specific genome modification (SSM) is an important tool for mosquito functional genomics and comparative gene expression studies, which contribute to a better understanding of mosquito biology and are thus a key to finding new strategies to eliminate vector-borne diseases. Moreover, it allows for the creation of advanced transgenic strains for vector control programs. SSM circumvents the drawbacks of transposon-mediated transgenesis, where random transgene integration into the host genome results in insertional mutagenesis and variable position effects. We applied the Cre/lox recombinase-mediated cassette exchange (RMCE) system to Aedes aegypti, the vector of dengue, chikungunya, and Zika viruses. In this context we created four target site lines for RMCE and evaluated their fitness costs. Cre-RMCE is functional in a two-step mechanism and with good efficiency in Ae. aegypti. The advantages of Cre-RMCE over existing site-specific modification systems for Ae. aegypti, phiC31-RMCE and CRISPR, originate in the preservation of the recombination sites, which 1) allows successive modifications and rapid expansion or adaptation of existing systems by repeated targeting of the same site; and 2) provides reversibility, thus allowing the excision of undesired sequences. Thereby, Cre-RMCE complements existing genomic modification tools, adding flexibility and versatility to vector genome targeting. PMID:28266580

  6. Estimating dengue vector abundance in the wet and dry season: implications for targeted vector control in urban and peri-urban Asia

    PubMed Central

    Wai, Khin Thet; Arunachalam, Natarajan; Tana, Susilowati; Espino, Fe; Kittayapong, Pattamaporn; Abeyewickreme, W; Hapangama, Dilini; Tyagi, Brij Kishore; Htun, Pe Than; Koyadun, Surachart; Kroeger, Axel; Sommerfeld, Johannes; Petzold, Max

    2012-01-01

    Background Research has shown that the classical Stegomyia indices (or “larval indices”) of the dengue vector Aedes aegypti reflect the absence or presence of the vector but do not provide accurate measures of adult mosquito density. In contrast, pupal indices as collected in pupal productivity surveys are a much better proxy indicator for adult vector abundance. However, it is unknown when it is most optimal to conduct pupal productivity surveys, in the wet or in the dry season or in both, to inform control services about the most productive water container types and if this pattern varies among different ecological settings. Methods A multi-country study in randomly selected twelve to twenty urban and peri-urban neighborhoods (“clusters”) of six Asian countries, in which all water holding containers were examined for larvae and pupae of Aedes aegypti during the dry season and the wet season and their productivity was characterized by water container types. In addition, meteorological data and information on reported dengue cases were collected. Findings The study reconfirmed the association between rainfall and dengue cases (“dengue season”) and underlined the importance of determining through pupal productivity surveys the “most productive containers types”, responsible for the majority (>70%) of adult dengue vectors. The variety of productive container types was greater during the wet than during the dry season, but included practically all container types productive in the dry season. Container types producing pupae were usually different from those infested by larvae indicating that containers with larval infestations do not necessarily foster pupal development and thus the production of adult Aedes mosquitoes. Conclusion Pupal productivity surveys conducted during the wet season will identify almost all of the most productive container types for both the dry and wet seasons and will therefore facilitate cost-effective targeted interventions

  7. Targeted delivery of biodegradable nanoparticles with ultrasound-targeted microbubble destruction-mediated hVEGF-siRNA transfection in human PC-3 cells in vitro.

    PubMed

    Li, Yun-Hua; Shi, Qiu-Sheng; Du, Jing; Jin, Li-Fang; Du, Lian-Fang; Liu, Pei-Feng; Duan, You-Rong

    2013-01-01

    A potentially viable approach for treating late-stage prostate cancer is gene therapy. Successful gene therapy requires safe and efficient delivery systems. In this study, we report the efficient delivery of small interfering RNA (siRNA) via the use of biodegradable nanoparticles (NPs) made from monomethoxypoly(ethylene glycol)-poly(lactic-co-glycolic acid)-poly-l-lysine (mPEG-PLGA-PLL) triblock copolymers. On the basis of previous findings, cyclic Arg-Gly-Asp (cRGD) peptides were conjugated to NPs to recognize the target site, integrin αvβ3, expressed in high levels in PC-3 prostate cancer cells. The suppression of angiogenesis by the downregulation of vascular endothelial growth factor (VEGF) expression has been widely used to inhibit the growth of malignant tumors. In our study, human VEGF (hVEGF)-siRNA was encapsulated in NPs to inhibit VEGF expression in PC-3 cells. Concurrently, sonoporation induced by ultrasound-targeted microbubble destruction (UTMD) was utilized for the delivery of siRNA-loaded NPs. Our results showed low cytotoxicity and high gene transfection efficiency, demonstrating that the targeted delivery of biodegradable NPs with UTMD may be potentially applied as new vector system for gene delivery.

  8. Multilayered polyion complexes with dissolvable silica layer covered by controlling densities of cRGD-conjugated PEG chains for cancer-targeted siRNA delivery.

    PubMed

    Naito, Mitsuru; Azuma, Ryota; Takemoto, Hiroyasu; Hori, Mao; Yoshinaga, Naoto; Osawa, Shigehito; Kamegawa, Rimpei; Kim, Hyun Jin; Ishii, Takehiko; Nishiyama, Nobuhiro; Miyata, Kanjiro; Kataoka, Kazunori

    2017-03-12

    Surface functionalization of nanoparticles is a crucial factor for nanoparticle-mediated drug and nucleic acid delivery. Particularly, the density of targeting ligands on nanoparticle significantly affects the affinity of nanoparticles to specific cellular surface (or receptor) through the multivalent binding effect. Herein, multilayered polyion complexes (mPICs) are prepared to possess varying densities of cyclic RGD peptide (cRGD) ligands for cancer-targeted small interfering RNA (siRNA) delivery. A template PIC is first prepared by mixing siRNAs with homo catiomers of N-substituted polyaspartamide bearing tetraethylenepentamine (PAsp(TEP)) in aqueous solution, followed by silica-coating through silicate condensation reaction. Then, silica-coated PICs (sPICs) are further covered with block catiomers of PAsp(TEP) and poly(ethylene glycol) (PEG) equipped with cRGD ligand. Successful preparation of targeted mPICs is confirmed from the changes in size and ζ-potential and the elemental analysis by transmission electron microscopy. Notably, the number of cRGD ligands per mPIC is regulated by altering the silicate concentration upon preparation of sPICs, which is confirmed by fluorescence correlation spectroscopy using fluorescent-labeled block catiomers. Ultimately, the targeted mPICs with a higher number of cRGD ligands demonstrate more efficient cellular uptake in cultured cancer cells, leading to enhanced gene silencing activity.

  9. Gene Therapy of Disseminated Breast Cancer Using Adenoviral Vectors Targeted Through Immunological Methods

    DTIC Science & Technology

    1998-08-01

    vectors encoding the firefly luciferase and 13-galactosidase reporter genes. In addition to these, an adenovirus vector encoding for the cytosine ... deaminase (CD) gene will be used to perform therapeutic studies. The CD enzyme converts the prodrug 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU), a

  10. Co-Development of Diagnostic Vectors to Support Targeted Therapies and Theranostics: Essential Tools in Personalized Cancer Therapy

    PubMed Central

    Nicolaides, Nicholas C.; O’Shannessy, Daniel J.; Albone, Earl; Grasso, Luigi

    2014-01-01

    potentially improving the therapeutic activity of novel, target-specific therapies that may otherwise have off-target toxicities or less efficacy in cells exhibiting certain pathways. Here, we discuss the use of co-developing diagnostic-targeting vectors to identify patients whose malignant tissue can specifically uptake a targeted anti-cancer drug vector prior to treatment. Using this system, a patient can be predetermined in real-time as to whether or not their tumor(s) can specifically uptake a drug-linked diagnostic vector, thus inferring the uptake of a similar vector linked to an anti-cancer agent. If tumor-specific uptake is observed, then the patient may be suitable for drug-linked vector therapy and have a higher likelihood of clinical benefit while patients with no tumor uptake should consider other therapeutic options. This approach offers complementary opportunities to rapidly develop broad tumor-specific agents for use in personalized medicine. PMID:24982846

  11. Computerized method for measurement of displacement vectors of target positions on EPID cine images in stereotactic radiotherapy

    NASA Astrophysics Data System (ADS)

    Arimura, Hidetaka; Anai, Shigeo; Yoshidome, Satoshi; Nakamura, Katsumasa; Shioyama, Yoshiyuki; Nomoto, Satoshi; Honda, Hiroshi; Onizuka, Yoshihiko; Terashima, Hiromi

    2007-03-01

    The purpose of this study was to develop a computerized method for measurement of displacement vectors of target position on electronic portal imaging device (EPID) cine images in a treatment without implanted markers. Our proposed method was based on a template matching technique with cross-correlation coefficient between a reference portal (RP) image and each consecutive portal (CP) image acquired by the EPID. EPID images with 512×384 pixels (pixel size:0.56 mm) were acquired in a cine mode at a sampling rate of 0.5 frame/sec by using an energy of 4, 6, or 10MV on linear accelerators. The displacement vector of the target on each cine image was determined from the position in which took the maximum cross-correlation value between the RP image and each CP image. We applied our method to EPID cine images of a lung phantom with a tumor model simulating respiratory motion, and 5 cases with a non-small cell lung cancer and one case of metastasis. For validation of our proposed method, displacement vectors of a target position calculated by our method were compared with those determined manually by two radiation oncologists. As a result, for lung phantom images, target displacements by our method correlated well with those by the oncologists (r=0.972 - 0.994). Correlation values for 4 cases ranged from 0.854 to 0.991, but the values for the other two cases were 0.609 and 0.644. This preliminary result suggested that our method may be useful for monitoring of displacement vectors of target positions without implanted markers in stereotactic radiotherapy.

  12. Identification of Adeno-Associated Viral Vectors That Target Neonatal and Adult Mammalian Inner Ear Cell Subtypes.

    PubMed

    Shu, Yilai; Tao, Yong; Wang, Zhengmin; Tang, Yong; Li, Huawei; Dai, Pu; Gao, Guangping; Chen, Zheng-Yi

    2016-09-01

    The mammalian inner ear consists of diverse cell types with important functions. Gene mutations in these diverse cell types have been found to underlie different forms of genetic hearing loss. Targeting these mutations for gene therapy development represents a future therapeutic strategy to treat hearing loss. Adeno-associated viral (AAV) vectors have become the vector of choice for gene delivery in animal models in vivo. To identify AAV vectors that target inner ear cell subtypes, we systemically screened 12 AAV vectors with different serotypes (AAV1, 2, 5, 6, 6.2, 7, 8, 9, rh.8, rh.10, rh.39, and rh.43) that carry a reporter gene GFP in neonatal and adult mice by microinjection in vivo. We found that most AAVs infect both neonatal and adult inner ear, with different specificities and expression levels. The inner ear cochlear sensory epithelial region, which includes auditory hair cells and supporting cells, is most frequently targeted for gene delivery. Expression of the transgene is sustained, and neonatal inner ear delivery does not adversely affect hearing. Adult inner ear injection of AAV has a similar infection pattern as the younger inner ear, with the exception that outer hair cell death caused by the injection procedure can lead to hearing loss. In the adult, more so than in the neonatal mice, cell types infected and efficiency of infection are correlated with the site of injection. Most infected cells survive in neonatal and adult inner ears. The study adds to the list of AAV vectors that transduce the mammalian inner ear efficiently, providing the tools that are important to study inner ear gene function and for the development of gene therapy to treat hearing loss.

  13. Identification of Adeno-Associated Viral Vectors That Target Neonatal and Adult Mammalian Inner Ear Cell Subtypes

    PubMed Central

    Shu, Yilai; Tao, Yong; Wang, Zhengmin; Tang, Yong; Li, Huawei; Dai, Pu; Gao, Guangping; Chen, Zheng-Yi

    2016-01-01

    The mammalian inner ear consists of diverse cell types with important functions. Gene mutations in these diverse cell types have been found to underlie different forms of genetic hearing loss. Targeting these mutations for gene therapy development represents a future therapeutic strategy to treat hearing loss. Adeno-associated viral (AAV) vectors have become the vector of choice for gene delivery in animal models in vivo. To identify AAV vectors that target inner ear cell subtypes, we systemically screened 12 AAV vectors with different serotypes (AAV1, 2, 5, 6, 6.2, 7, 8, 9, rh.8, rh.10, rh.39, and rh.43) that carry a reporter gene GFP in neonatal and adult mice by microinjection in vivo. We found that most AAVs infect both neonatal and adult inner ear, with different specificities and expression levels. The inner ear cochlear sensory epithelial region, which includes auditory hair cells and supporting cells, is most frequently targeted for gene delivery. Expression of the transgene is sustained, and neonatal inner ear delivery does not adversely affect hearing. Adult inner ear injection of AAV has a similar infection pattern as the younger inner ear, with the exception that outer hair cell death caused by the injection procedure can lead to hearing loss. In the adult, more so than in the neonatal mice, cell types infected and efficiency of infection are correlated with the site of injection. Most infected cells survive in neonatal and adult inner ears. The study adds to the list of AAV vectors that transduce the mammalian inner ear efficiently, providing the tools that are important to study inner ear gene function and for the development of gene therapy to treat hearing loss. PMID:27342665

  14. Development of a Nature-Inspired Vector for Targeted Systemic Breast Cancer Gene Therapy

    DTIC Science & Technology

    2009-01-01

    original design by DNA sequencing. The pET21b:DBV expression vector was transformed into E . coli BL21(DE3) pLysS. Starter cultures, 5 ml, were...cloning process. The expression system was transformed into E . coli BL21 (DE3) plysS and DBV was expressed and purified at a 2 mg/liter yield. The...vectors: The genes encoding various motifs were synthesized and cloned  into a pET21b expression vector. The expression vector was transformed into  E

  15. A Hypoxia-Responsive Glial Cell–Specific Gene Therapy Vector for Targeting Retinal Neovascularization

    PubMed Central

    Biswal, Manas R.; Prentice, Howard M.; Dorey, C. Kathleen; Blanks, Janet C.

    2014-01-01

    Purpose. Müller cells, the major glial cell in the retina, play a significant role in retinal neovascularization in response to tissue hypoxia. We previously designed and tested a vector using a hypoxia-responsive domain and a glial fibrillary acidic protein (GFAP) promoter to drive green fluorescent protein (GFP) expression in Müller cells in the murine model of oxygen-induced retinopathy (OIR). This study compares the efficacy of regulated and unregulated Müller cell delivery of endostatin in preventing neovascularization in the OIR model. Methods. Endostatin cDNA was cloned into plasmids with hypoxia-regulated GFAP or unregulated GFAP promoters, and packaged into self-complementary adeno-associated virus serotype 2 vectors (scAAV2). Before placement in hyperoxia on postnatal day (P)7, mice were given intravitreal injections of regulated or unregulated scAAV2, capsid, or PBS. Five days after return to room air, on P17, neovascular and avascular areas, as well as expression of the transgene and vascular endothelial growth factor (VEGF), were compared in OIR animals treated with a vector, capsid, or PBS. Results. The hypoxia-regulated, glial-specific, vector-expressing endostatin reduced neovascularization by 93% and reduced the central vaso-obliteration area by 90%, matching the results with the unregulated GFAP-Endo vector. Retinas treated with the regulated endostatin vector expressed substantial amounts of endostatin protein, and significantly reduced VEGF protein. Endostatin production from the regulated vector was undetectable in retinas with undamaged vasculature. Conclusions. These findings suggest that the hypoxia-regulated, glial cell–specific vector expressing endostatin may be useful for treatment of neovascularization in proliferative diabetic retinopathy. PMID:25377223

  16. Development of a Nature-Inspired Vector for Targeted Systemic Breast Cancer Gene Therapy

    DTIC Science & Technology

    2008-09-01

    The mass spectroscopy confirmed the size of the vectors which was in agreement with the expected theoretical values. The cathepsin D substrate...16% was obtained at a Vector to DNA ratio of 10 as detected by flowcytometry. For more details, please see Appendix A. Taking advantage of...structure we have utilized arginine rich adenovirus µ peptide [18] to 4 complex with pDNA and form condensed nanosize particles. To examine the extent

  17. Photoreceptor-targeted gene delivery using intravitreally administered AAV vectors in dogs

    PubMed Central

    Boyd, RF; Sledge, DG; Boye, SL; Boye, SE; Hauswirth, WW; Komáromy, AM; Petersen-Jones, SM; Bartoe, JT

    2016-01-01

    Delivery of therapeutic transgenes to retinal photoreceptors using adeno-associated virus (AAV) vectors has traditionally required subretinal injection. Recently, photoreceptor transduction efficiency following intravitreal injection (IVT) has improved in rodent models through use of capsid-mutant AAV vectors; but remains limited in large animal models. Thickness of the inner limiting membrane (ILM) in large animals is thought to impair retinal penetration by AAV. Our study compared two newly developed AAV vectors containing multiple capsid amino acid substitutions following IVT in dogs. The ability of two promoter constructs to restrict reporter transgene expression to photoreceptors was also evaluated. AAV vectors containing the interphotoreceptor-binding protein (IRBP) promoter drove expression exclusively in rod and cone photoreceptors, with transduction efficiencies of ~ 4% of cones and 2% of rods. Notably, in the central region containing the cone-rich visual streak, 15.6% of cones were transduced. Significant regional variation existed, with lower transduction efficiencies in the temporal regions of all eyes. This variation did not correlate with ILM thickness. Vectors carrying a cone-specific promoter failed to transduce a quantifiable percentage of cone photoreceptors. The newly developed AAV vectors containing the IRBP promoter were capable of producing photoreceptor-specific transgene expression following IVT in the dog. PMID:26467396

  18. Photoreceptor-targeted gene delivery using intravitreally administered AAV vectors in dogs.

    PubMed

    Boyd, R F; Sledge, D G; Boye, S L; Boye, S E; Hauswirth, W W; Komáromy, A M; Petersen-Jones, S M; Bartoe, J T

    2016-02-01

    Delivery of therapeutic transgenes to retinal photoreceptors using adeno-associated virus (AAV) vectors has traditionally required subretinal injection. Recently, photoreceptor transduction efficiency following intravitreal injection (IVT) has improved in rodent models through use of capsid-mutant AAV vectors; but remains limited in large animal models. Thickness of the inner limiting membrane (ILM) in large animals is thought to impair retinal penetration by AAV. Our study compared two newly developed AAV vectors containing multiple capsid amino acid substitutions following IVT in dogs. The ability of two promoter constructs to restrict reporter transgene expression to photoreceptors was also evaluated. AAV vectors containing the interphotoreceptor-binding protein (IRBP) promoter drove expression exclusively in rod and cone photoreceptors, with transduction efficiencies of ~4% of cones and 2% of rods. Notably, in the central region containing the cone-rich visual streak, 15.6% of cones were transduced. Significant regional variation existed, with lower transduction efficiencies in the temporal regions of all eyes. This variation did not correlate with ILM thickness. Vectors carrying a cone-specific promoter failed to transduce a quantifiable percentage of cone photoreceptors. The newly developed AAV vectors containing the IRBP promoter were capable of producing photoreceptor-specific transgene expression following IVT in the dog.

  19. A multicolor panel of TALE-KRAB based transcriptional repressor vectors enabling knockdown of multiple gene targets

    PubMed Central

    Zhang, Zhonghui; Wu, Elise; Qian, Zhijian; Wu, Wen-Shu

    2014-01-01

    Stable and efficient knockdown of multiple gene targets is highly desirable for dissection of molecular pathways. Because it allows sequence-specific DNA binding, transcription activator-like effector (TALE) offers a new genetic perturbation technique that allows for gene-specific repression. Here, we constructed a multicolor lentiviral TALE-Kruppel-associated box (KRAB) expression vector platform that enables knockdown of multiple gene targets. This platform is fully compatible with the Golden Gate TALEN and TAL Effector Kit 2.0, a widely used and efficient method for TALE assembly. We showed that this multicolor TALE-KRAB vector system when combined together with bone marrow transplantation could quickly knock down c-kit and PU.1 genes in hematopoietic stem and progenitor cells of recipient mice. Furthermore, our data demonstrated that this platform simultaneously knocked down both c-Kit and PU.1 genes in the same primary cell populations. Together, our results suggest that this multicolor TALE-KRAB vector platform is a promising and versatile tool for knockdown of multiple gene targets and could greatly facilitate dissection of molecular pathways. PMID:25475013

  20. Targeted, homology-driven gene insertion in stem cells by ZFN-loaded ‘all-in-one’ lentiviral vectors

    PubMed Central

    Cai, Yujia; Laustsen, Anders; Zhou, Yan; Sun, Chenglong; Anderson, Mads Valdemar; Li, Shengting; Uldbjerg, Niels; Luo, Yonglun; Jakobsen, Martin R; Mikkelsen, Jacob Giehm

    2016-01-01

    Biased integration remains a key challenge for gene therapy based on lentiviral vector technologies. Engineering of next-generation lentiviral vectors targeting safe genomic harbors for insertion is therefore of high relevance. In a previous paper (Cai et al., 2014a), we showed the use of integrase-defective lentiviral vectors (IDLVs) as carriers of complete gene repair kits consisting of zinc-finger nuclease (ZFN) proteins and repair sequences, allowing gene correction by homologous recombination (HR). Here, we follow this strategy to engineer ZFN-loaded IDLVs that insert transgenes by a homology-driven mechanism into safe loci. This insertion mechanism is driven by time-restricted exposure of treated cells to ZFNs. We show targeted gene integration in human stem cells, including CD34+ hematopoietic progenitors and induced pluripotent stem cells (iPSCs). Notably, targeted insertions are identified in 89% of transduced iPSCs. Our findings demonstrate the applicability of nuclease-loaded ‘all-in-one’ IDLVs for site-directed gene insertion in stem cell-based gene therapies. DOI: http://dx.doi.org/10.7554/eLife.12213.001 PMID:27278774

  1. Peptide-Mediated Tumor Targeting by a Degradable Nano Gene Delivery Vector Based on Pluronic-Modified Polyethylenimine

    NASA Astrophysics Data System (ADS)

    Wu, Zhaoyong; Zhan, Shuyu; Fan, Wei; Ding, Xueying; Wu, Xin; Zhang, Wei; Fu, Yinghua; Huang, Yueyan; Huang, Xuan; Chen, Rubing; Li, Mingjuan; Xu, Ningyin; Zheng, Yongxia; Ding, Baoyue

    2016-03-01

    Polyethylenimine (PEI) is considered to be a promising non-viral gene delivery vector. To solve the toxicity versus efficacy and tumor-targeting challenges of PEI used as gene delivery vector, we constructed a novel non-viral vector DR5-TAT-modified Pluronic-PEI (Pluronic-PEI-DR5-TAT), which was based on the attachment of low-molecular-weight polyethylenimine (LMW-PEI) to the amphiphilic polymer Pluronic to prepare Pluronic-modified LMW-PEI (Pluronic-PEI). This was then conjugated to a multifunctional peptide containing a cell-penetrating peptide (TAT) and a synthetic peptide that would bind to DR5—a receptor that is overexpressed in cancer cells. The vector showed controlled degradation, favorable DNA condensation and protection performance. The Pluronic-PEI-DR5-TAT/DNA complexes at an N/P ratio of 15:1 were spherical nanoparticles of 122 ± 11.6 nm and a zeta potential of about 22 ± 2.8 mV. In vitro biological characterization results indicated that Pluronic-PEI-DR5-TAT/DNA complexes had a higher specificity for the DR5 receptor and were taken up more efficiently by tumor cells than normal cells, compared to complexes formed with PEI 25 kDa or Pluronic-PEI. Thus, the novel complexes showed much lower cytotoxicity to normal cells and higher gene transfection efficiency in tumor cells than that exhibited by PEI 25 kDa and Pluronic-PEI. In summary, our novel, degradable non-viral tumor-targeting vector is a promising candidate for use in gene therapy.

  2. Differential targeting of feline photoreceptors by recombinant adeno-associated viral vectors: implications for preclinical gene therapy trials.

    PubMed

    Minella, A L; Mowat, F M; Willett, K L; Sledge, D; Bartoe, J T; Bennett, J; Petersen-Jones, S M

    2014-10-01

    The cat is emerging as a promising large animal model for preclinical testing of retinal dystrophy therapies, for example, by gene therapy. However, there is a paucity of studies investigating viral vector gene transfer to the feline retina. We therefore sought to study the tropism of recombinant adeno-associated viral (rAAV) vectors for the feline outer retina. We delivered four rAAV serotypes: rAAV2/2, rAAV2/5, rAAV2/8 and rAAV2/9, each expressing green fluorescent protein (GFP) under the control of a cytomegalovirus promoter, to the subretinal space in cats and, for comparison, mice. Cats were monitored for gene expression by in vivo imaging and cellular tropism was determined using immunohistochemistry. In cats, rAAV2/2, rAAV2/8 and rAAV2/9 vectors induced faster and stronger GFP expression than rAAV2/5 and all vectors transduced the retinal pigment epithelium (RPE) and photoreceptors. Unlike in mice, cone photoreceptors in the cat retina were more efficiently transduced than rod photoreceptors. In mice, rAAV2/2 only transduced the RPE whereas the other vectors also transduced rods and cones. These results highlight species differences in cellular tropism of rAAV vectors in the outer retina. We conclude that rAAV serotypes are suitable for use for retinal gene therapy in feline models, particularly when cone photoreceptors are the target cell.

  3. A single-plasmid vector for transgene amplification using short hairpin RNA targeting the 3'-UTR of amplifiable dhfr.

    PubMed

    Kang, Shin-Young; Kim, Yeon-Gu; Lee, Hong Weon; Lee, Eun Gyo

    2015-12-01

    Gene amplification using dihydrofolate reductase gene (dhfr) and methotrexate (MTX) is widely used for recombinant protein production in mammalian cells and is typically conducted in DHFR-deficient Chinese hamster ovary (CHO) cell lines. Generation of DHFR-deficient cells can be achieved by an expression vector incorporating short hairpin RNA (shRNA) that targets the 3'-untranslated region (UTR) of endogenous dhfr. Thus, shRNAs were designed to target the 3'-UTR of endogenous dhfr, and shRNA-2 efficiently down-regulated dhfr expression in CHO-K1 cells. A single gene copy of shRNA-2 also decreased the translational level of DHFR by 80% in Flp-In CHO cells. shRNA-2 was then incorporated into a plasmid vector expressing human erythropoietin (EPO) and an exogenous DHFR to develop EPO-producing cells in the Flp-In system. The specific EPO productivity (q EPO) was enhanced by stepwise increments of MTX concentration, and differences in the amplification rate were observed in Flp-In CHO cells that expressed shRNA-2. In addition, the q EPO increased by more than 2.5-fold in the presence of 500 nM MTX. The mRNA expression level and gene copy numbers of dhfr were correlated with increased productivity in the cells, which is influenced by inhibition of endogenous dhfr. This study reveals that an expression vector including shRNA that targets the 3'-UTR of endogenous dhfr can enhance the transgene amplification rate and productivity by generating DHFR-deficient cells. This approach may be applied for amplifying the foreign gene in wild-type cell lines as a versatile single-plasmid vector.

  4. Combinatorial support vector machines approach for virtual screening of selective multi-target serotonin reuptake inhibitors from large compound libraries.

    PubMed

    Shi, Z; Ma, X H; Qin, C; Jia, J; Jiang, Y Y; Tan, C Y; Chen, Y Z

    2012-02-01

    Selective multi-target serotonin reuptake inhibitors enhance antidepressant efficacy. Their discovery can be facilitated by multiple methods, including in silico ones. In this study, we developed and tested an in silico method, combinatorial support vector machines (COMBI-SVMs), for virtual screening (VS) multi-target serotonin reuptake inhibitors of seven target pairs (serotonin transporter paired with noradrenaline transporter, H(3) receptor, 5-HT(1A) receptor, 5-HT(1B) receptor, 5-HT(2C) receptor, melanocortin 4 receptor and neurokinin 1 receptor respectively) from large compound libraries. COMBI-SVMs trained with 917-1951 individual target inhibitors correctly identified 22-83.3% (majority >31.1%) of the 6-216 dual inhibitors collected from literature as independent testing sets. COMBI-SVMs showed moderate to good target selectivity in misclassifying as dual inhibitors 2.2-29.8% (majority <15.4%) of the individual target inhibitors of the same target pair and 0.58-7.1% of the other 6 targets outside the target pair. COMBI-SVMs showed low dual inhibitor false hit rates (0.006-0.056%, 0.042-0.21%, 0.2-4%) in screening 17 million PubChem compounds, 168,000 MDDR compounds, and 7-8181 MDDR compounds similar to the dual inhibitors. Compared with similarity searching, k-NN and PNN methods, COMBI-SVM produced comparable dual inhibitor yields, similar target selectivity, and lower false hit rate in screening 168,000 MDDR compounds. The annotated classes of many COMBI-SVMs identified MDDR virtual hits correlate with the reported effects of their predicted targets. COMBI-SVM is potentially useful for searching selective multi-target agents without explicit knowledge of these agents.

  5. Transcriptional Targeting of Mature Dendritic Cells with Adenoviral Vectors via a Modular Promoter System for Antigen Expression and Functional Manipulation

    PubMed Central

    Deinzer, Andrea

    2016-01-01

    To specifically target dendritic cells (DCs) to simultaneously express different therapeutic transgenes for inducing immune responses against tumors, we used a combined promoter system of adenoviral vectors. We selected a 216 bp short Hsp70B′ core promoter induced by a mutated, constitutively active heat shock factor (mHSF) 1 to drive strong gene expression of therapeutic transgenes MelanA, BclxL, and IL-12p70 in HeLa cells, as well as in mature DCs (mDCs). As this involves overexpressing mHSF1, we first evaluated the resulting effects on DCs regarding upregulation of heat shock proteins and maturation markers, toxicity, cytokine profile, and capacity to induce antigen-specific CD8+ T cells. Second, we generated the two-vector-based “modular promoter” system, where one vector contains the mHSF1 under the control of the human CD83 promoter, which is specifically active only in DCs and after maturation. mHSF1, in turn, activates the Hsp70B′ core promotor-driven expression of transgenes MelanA and IL-12p70 in the DC-like cell line XS52 and in human mature and hence immunogenic DCs, but not in tolerogenic immature DCs. These in vitro experiments provide the basis for an in vivo targeting of mature DCs for the expression of multiple transgenes. Therefore, this modular promoter system represents a promising tool for future DC-based immunotherapies in vivo. PMID:27446966

  6. Elevation of intraocular pressure in rodents using viral vectors targeting the trabecular meshwork.

    PubMed

    Pang, Iok-Hou; Millar, J Cameron; Clark, Abbot F

    2015-12-01

    Rodents are increasingly being used as glaucoma models to study ocular hypertension, optic neuropathy, and retinopathy. A number of different techniques are used to elevate intraocular pressure in rodent eyes by artificially obstructing the aqueous outflow pathway. Another successful technique to induce ocular hypertension is to transduce the trabecular meshwork of rodent eyes with viral vectors expressing glaucoma associated transgenes to provide more relevant models of glaucomatous damage to the trabecular meshwork. This technique has been used to validate newly discovered glaucoma pathogenesis pathways as well as to develop rodent models of primary open angle glaucoma. Ocular hypertension has successfully been induced by adenovirus 5 mediated delivery of mutant MYOC, bioactivated TGFβ2, SFRP1, DKK1, GREM1, and CD44. Advantages of this approach are: selective tropism for the trabecular meshwork, the ability to use numerous mouse strains, and the relatively rapid onset of IOP elevation. Disadvantages include mild-to-moderate ocular inflammation induced by the Ad5 vector and sometimes transient transgene expression. Current efforts are focused at discovering less immunogenic viral vectors that have tropism for the trabecular meshwork and drive sufficient transgene expression to induce ocular hypertension. This viral vector approach allows rapid proof of concept studies to study glaucomatous damage to the trabecular meshwork without the expensive and time-consuming generation of transgenic mouse lines.

  7. Self-focusing therapeutic gene delivery with intelligent gene vector swarms: intra-swarm signalling through receptor transgene expression in targeted cells.

    PubMed

    Tolmachov, Oleg E

    2015-01-01

    Gene delivery in vivo that is tightly focused on the intended target cells is essential to maximize the benefits of gene therapy and to reduce unwanted side-effects. Cell surface markers are immediately available for probing by therapeutic gene vectors and are often used to direct gene transfer with these vectors to specific target cell populations. However, it is not unusual for the choice of available extra-cellular markers to be too scarce to provide a reliable definition of the desired therapeutically relevant set of target cells. Therefore, interrogation of intra-cellular determinants of cell-specificity, such as tissue-specific transcription factors, can be vital in order to provide detailed cell-guiding information to gene vector particles. An important improvement in cell-specific gene delivery can be achieved through auto-buildup in vector homing efficiency using intelligent 'self-focusing' of swarms of vector particles on target cells. Vector self-focusing was previously suggested to rely on the release of diffusible chemo-attractants after a successful target-specific hit by 'scout' vector particles. I hypothesize that intelligent self-focusing behaviour of swarms of cell-targeted therapeutic gene vectors can be accomplished without the employment of difficult-to-use diffusible chemo-attractants, instead relying on the intra-swarm signalling through cells expressing a non-diffusible extra-cellular receptor for the gene vectors. In the proposed model, cell-guiding information is gathered by the 'scout' gene vector particles, which: (1) attach to a variety of cells via a weakly binding (low affinity) receptor; (2) successfully facilitate gene transfer into these cells; (3) query intra-cellular determinants of cell-specificity with their transgene expression control elements and (4) direct the cell-specific biosynthesis of a vector-encoded strongly binding (high affinity) cell-surface receptor. Free members of the vector swarm loaded with therapeutic cargo

  8. [New protein vectors based on an alpha-fetoprotein fragment for targeted DNA delivery into cancer cells].

    PubMed

    Tatarinova, O N; Gorokhovets, N V; Makarov, V A; Posypanova, G A; Serebriakova, M V; Pozmogova, G E

    2010-01-01

    A human alpha-fetoprotein fragment (AFP) modified with oligocationic homologs of nuclear localization signal was used to construct new target cell-selective DNA-carrier proteins. The new recombinant vectors containing C- or N-terminal polynucleotide-binding domains are able to form stable complexes with single- or double-stranded oligonucleotides and plasmid DNA. Using flow cytometry and fluorescent microscopy, it was shown that such nucleoprotein complexes can be selectively internalized in target cells receptors superexpressing AFP receptors. The results obtained are important both for understanding mechanisms of formation of DNA-protein complexes and for studying their interaction with intracellular molecular targets. The new proteins can be used as a tool for the development of highly selective and efficacious gene-selective antitumour drugs.

  9. Analytical derivation of distortion constraints and their verification in a learning vector quantization-based target recognition system

    NASA Astrophysics Data System (ADS)

    Iftekharuddin, Khan M.; Razzaque, Mohammad A.

    2005-06-01

    We obtain a novel analytical derivation for distortion-related constraints in a neural network- (NN)-based automatic target recognition (ATR) system. We obtain two types of constraints for a realistic ATR system implementation involving 4-f correlator architecture. The first constraint determines the relative size between the input objects and input correlation filters. The second constraint dictates the limits on amount of rotation, translation, and scale of input objects for system implementation. We exploit these constraints in recognition of targets varying in rotation, translation, scale, occlusion, and the combination of all of these distortions using a learning vector quantization (LVQ) NN. We present the simulation verification of the constraints using both the gray-scale images and Defense Advanced Research Projects Agency's (DARPA's) Moving and Stationary Target Recognition (MSTAR) synthetic aperture radar (SAR) images with different depression and pose angles.

  10. Novel viral vectors utilizing intron splice-switching to activate genome rescue, expression and replication in targeted cells

    PubMed Central

    2011-01-01

    Background The outcome of virus infection depends from the precise coordination of viral gene expression and genome replication. The ability to control and regulate these processes is therefore important for analysis of infection process. Viruses are also useful tools in bio- and gene technology; they can efficiently kill cancer cells and trigger immune responses to tumors. However, the methods for constructing tissue- or cell-type specific viruses typically suffer from low target-cell specificity and a high risk of reversion. Therefore novel and universal methods of regulation of viral infection are also important for therapeutic application of virus-based systems. Methods Aberrantly spliced introns were introduced into crucial gene-expression units of adenovirus vector and alphavirus DNA/RNA layered vectors and their effects on the viral gene expression, replication and/or the release of infectious genomes were studied in cell culture. Transfection of the cells with splice-switching oligonucleotides was used to correct the introduced functional defect(s). Results It was demonstrated that viral gene expression, replication and/or the release of infectious genomes can be blocked by the introduction of aberrantly spliced introns. The insertion of such an intron into an adenovirus vector reduced the expression of the targeted gene more than fifty-fold. A similar insertion into an alphavirus DNA/RNA layered vector had a less dramatic effect; here, only the release of the infectious transcript was suppressed but not the subsequent replication and spread of the virus. However the insertion of two aberrantly spliced introns resulted in an over one hundred-fold reduction in the infectivity of the DNA/RNA layered vector. Furthermore, in both systems the observed effects could be reverted by the delivery of splice-switching oligonucleotide(s), which corrected the splicing defects. Conclusions Splice-switch technology, originally developed for genetic disease therapy, can

  11. Promoters and serotypes: targeting of adeno-associated virus vectors for gene transfer in the rat central nervous system in vitro and in vivo.

    PubMed

    Shevtsova, Z; Malik, J M I; Michel, U; Bähr, M; Kügler, S

    2005-01-01

    The brain parenchyma consists of several different cell types, such as neurones, astrocytes, microglia, oligodendroglia and epithelial cells, which are morphologically and functionally intermingled in highly complex three-dimensional structures. These different cell types are also present in cultures of brain cells prepared to serve as model systems of CNS physiology. Gene transfer, either in a therapeutic attempt or in basic research, is a fascinating and promising tool to manipulate both the complex physiology of the brain and that of isolated neuronal cells. Viral vectors based on the parvovirus, adeno-associated virus (AAV), have emerged as powerful transgene delivery vehicles. Here we describe highly efficient targeting of AAV vectors to either neurones or astrocytes in cultured primary brain cell cultures. We also show that transcriptional targeting can be achieved by the use of small promoters, significantly boosting the transgene capacity of the recombinant viral genome. However, we also demonstrate that successful targeting of a vector in vitro does not necessarily imply that the same targeting works in the adult brain. Cross-packaging the AAV-2 genome in capsids of other serotypes adds additional benefits to this vector system. In the brain, the serotype-5 capsid allows for drastically increased spread of the recombinant vector as compared to the serotype-2 capsid. Finally, we emphasize the optimal targeting approach, in which the natural tropism of a vector for a specific cell type is employed. Taken together, these data demonstrate the flexibility which AAV-based vector systems offer in physiological research.

  12. Targeting B16 tumors in vivo with peptide-conjugated gold nanoparticles

    NASA Astrophysics Data System (ADS)

    Poon, Wilson; Zhang, Xuan; Bekah, Devesh; Teodoro, Jose G.; Nadeau, Jay L.

    2015-07-01

    This study examines the effects of polyethylene glycol (PEG) and peptide conjugation on the biodistribution of ultrasmall (2.7 nm) gold nanoparticles in mice bearing B16 melanoma allografts. Nanoparticles were delivered intravenously, and biodistribution was measured at specific timepoints by organ digestion and inductively coupled plasma mass spectrometry. All major organs were examined. Two peptides were tested: the cyclic RGD peptide (cRGD, which targets integrins); and a recently described peptide derived from the myxoma virus. We found the greatest specific tumor delivery using the myxoma peptide, with or without PEGylation. Un-PEGylated cRGD performed poorly, but PEGylated RGD showed a significant transient collection in the tumor. Liver and kidney were the primary targets of all constructs. None of the particles were able to cross the blood-brain barrier. Although it was able to deliver Au to B16 cells, the myxoma peptide did not show any cytotoxic activity against these cells, in contrast to previous reports. These results indicate that the effect of passive targeting by PEGylation and active targeting by peptides can be independent or combined, and that they should be evaluated on a case-by-case basis when designing new nanosystems for targeted therapies. Both myxoma peptide and cRGD should be considered for specific targeting to melanoma, but a thorough investigation of the cytotoxicity of the myxoma peptide to different cell lines remains to be performed.

  13. Gene therapy for cardiovascular disease: advances in vector development, targeting, and delivery for clinical translation.

    PubMed

    Rincon, Melvin Y; VandenDriessche, Thierry; Chuah, Marinee K

    2015-10-01

    Gene therapy is a promising modality for the treatment of inherited and acquired cardiovascular diseases. The identification of the molecular pathways involved in the pathophysiology of heart failure and other associated cardiac diseases led to encouraging preclinical gene therapy studies in small and large animal models. However, the initial clinical results yielded only modest or no improvement in clinical endpoints. The presence of neutralizing antibodies and cellular immune responses directed against the viral vector and/or the gene-modified cells, the insufficient gene expression levels, and the limited gene transduction efficiencies accounted for the overall limited clinical improvements. Nevertheless, further improvements of the gene delivery technology and a better understanding of the underlying biology fostered renewed interest in gene therapy for heart failure. In particular, improved vectors based on emerging cardiotropic serotypes of the adeno-associated viral vector (AAV) are particularly well suited to coax expression of therapeutic genes in the heart. This led to new clinical trials based on the delivery of the sarcoplasmic reticulum Ca(2+)-ATPase protein (SERCA2a). Though the first clinical results were encouraging, a recent Phase IIb trial did not confirm the beneficial clinical outcomes that were initially reported. New approaches based on S100A1 and adenylate cyclase 6 are also being considered for clinical applications. Emerging paradigms based on the use of miRNA regulation or CRISPR/Cas9-based genome engineering open new therapeutic perspectives for treating cardiovascular diseases by gene therapy. Nevertheless, the continuous improvement of cardiac gene delivery is needed to allow the use of safer and more effective vector doses, ultimately bringing gene therapy for heart failure one step closer to reality.

  14. Gene therapy for cardiovascular disease: advances in vector development, targeting, and delivery for clinical translation

    PubMed Central

    Rincon, Melvin Y.; VandenDriessche, Thierry; Chuah, Marinee K.

    2015-01-01

    Gene therapy is a promising modality for the treatment of inherited and acquired cardiovascular diseases. The identification of the molecular pathways involved in the pathophysiology of heart failure and other associated cardiac diseases led to encouraging preclinical gene therapy studies in small and large animal models. However, the initial clinical results yielded only modest or no improvement in clinical endpoints. The presence of neutralizing antibodies and cellular immune responses directed against the viral vector and/or the gene-modified cells, the insufficient gene expression levels, and the limited gene transduction efficiencies accounted for the overall limited clinical improvements. Nevertheless, further improvements of the gene delivery technology and a better understanding of the underlying biology fostered renewed interest in gene therapy for heart failure. In particular, improved vectors based on emerging cardiotropic serotypes of the adeno-associated viral vector (AAV) are particularly well suited to coax expression of therapeutic genes in the heart. This led to new clinical trials based on the delivery of the sarcoplasmic reticulum Ca2+-ATPase protein (SERCA2a). Though the first clinical results were encouraging, a recent Phase IIb trial did not confirm the beneficial clinical outcomes that were initially reported. New approaches based on S100A1 and adenylate cyclase 6 are also being considered for clinical applications. Emerging paradigms based on the use of miRNA regulation or CRISPR/Cas9-based genome engineering open new therapeutic perspectives for treating cardiovascular diseases by gene therapy. Nevertheless, the continuous improvement of cardiac gene delivery is needed to allow the use of safer and more effective vector doses, ultimately bringing gene therapy for heart failure one step closer to reality. PMID:26239654

  15. Alleviation of off-target effects from vector-encoded shRNAs via codelivered RNA decoys.

    PubMed

    Mockenhaupt, Stefan; Grosse, Stefanie; Rupp, Daniel; Bartenschlager, Ralf; Grimm, Dirk

    2015-07-28

    Exogenous RNAi triggers such as shRNAs ideally exert their activities exclusively via the antisense strand that binds and silences designated target mRNAs. However, in principle, the sense strand also possesses silencing capacity that may contribute to adverse RNAi side effects including off-target gene regulation. Here, we address this concern with a novel strategy that reduces sense strand activity of vector-encoded shRNAs via codelivery of inhibitory tough decoy (TuD) RNAs. Using various shRNAs for proof of concept, we validate that coexpression of TuDs can sequester and inactivate shRNA sense strands in human cells selectively without affecting desired antisense activities from the same shRNAs. Moreover, we show how coexpressed TuDs can alleviate shRNA-mediated perturbation of global gene expression by specifically de-repressing off-target transcripts carrying seed matches to the shRNA sense strand. Our combination of shRNA and TuD in a single bicistronic gene transfer vector derived from Adeno-associated virus (AAV) enables a wide range of applications, including gene therapies. To this end, we engineered our constructs in a modular fashion and identified simple hairpin design rules permitting adaptation to preexisting or new shRNAs. Finally, we demonstrate the power of our vectors for combinatorial RNAi strategies by showing robust suppression of hepatitis C virus (HCV) with an AAV expressing a bifunctional TuD against an anti-HCV shRNA sense strand and an HCV-related cellular miRNA. The data and tools reported here represent an important step toward the next generation of RNAi triggers with increased specificity and thus ultimately safety in humans.

  16. Singular vector based targeted observations of chemical constituents: description and first application of the EURAD-IM-SVA

    NASA Astrophysics Data System (ADS)

    Goris, N.; Elbern, H.

    2015-08-01

    Measurements of the large dimensional chemical state of the atmosphere provide only sparse snapshots of the state of the system due to their typically insufficient temporal and spatial density. In order to optimize the measurement configurations despite those limitations, the present work describes the identification of sensitive states of the chemical system as optimal target areas for adaptive observations. For this purpose, the technique of singular vector analysis (SVA), which has been proved effective for targeted observations in numerical weather predication, is implemented into the chemical transport model EURAD-IM (EURopean Air pollution and Dispersion - Inverse Model) yielding the EURAD-IM-SVA. Besides initial values, emissions are investigated as critical simulation controlling targeting variables. For both variants, singular vectors are applied to determine the optimal placement for observations and moreover to quantify which chemical compounds have to be observed with preference. Based on measurements of the airship based ZEPTER-2 campaign, the EURAD-IM-SVA has been evaluated by conducting a comprehensive set of model runs involving different initial states and simulation lengths. Since the considered cases are restricted in terms of considered chemical compounds and selected areas, they allow for a retracing of the results and a confirmation of their correctness. Our analysis shows that the optimal placement for observations of chemical species is not entirely determined by mere transport and mixing processes. Rather, a combination of initial chemical concentrations, chemical conversions, and meteorological processes determine the influence of chemical compounds and regions. We furthermore demonstrate that the optimal placement of observations of emission strengths is highly dependent on the location of emission sources and that the benefit of including emissions as target variables outperforms the value of initial value optimisation with growing

  17. MAC-T cells as a tool to evaluate lentiviral vector construction targeting recombinant protein expression in milk.

    PubMed

    Monzani, Paulo S; Guemra, Samuel; Adona, Paulo R; Ohashi, Otavio M; Meirelles, Flávio V; Wheeler, Matthew B

    2015-01-01

    Prior to generating transgenic animals for bioreactors, it is important to evaluate the vector constructed to avoid poor protein expression. Mammary epithelial cells cultured in vitro have been proposed as a model to reproduce the biology of the mammary gland. In the present work, three lentiviral vectors were constructed for the human growth hormone (GH), interleukin 2 (IL2), and granulocyte colony-stimulating factor 3 (CSF3) genes driven by the bovine β-casein promoter. The lentiviruses were used to transduce mammary epithelial cells (MAC-T), and the transformed cells were cultured on polystyrene in culture medium with and without prolactin. The gene expression of transgenes was evaluated by PCR using cDNA, and recombinant protein expression was evaluated by Western-blotting using concentrated medium and cellular extracts. The gene expression, of the three introduced genes, was detected in both induced and non induced MAC-T cells. The human GH protein was detected in the concentrated medium, whereas CSF3 was detected in the cellular extract. Apparently, the cellular extract is more appropriate than the concentrated medium to detect recombinant protein, principally because concentrated medium has a high concentration of bovine serum albumin. The results suggest that MAC-T cells may be a good system to evaluate vector construction targeting recombinant protein expression in milk.

  18. Preventive and curative effects of Apple latent spherical virus vectors harboring part of the target virus genome against potyvirus and cucumovirus infections.

    PubMed

    Tamura, Akihiro; Kato, Takahiro; Taki, Ayano; Sone, Mikako; Satoh, Nozomi; Yamagishi, Noriko; Takahashi, Tsubasa; Ryo, Bo-Song; Natsuaki, Tomohide; Yoshikawa, Nobuyuki

    2013-11-01

    Apple latent spherical virus (ALSV)-based vectors experimentally infect a broad range of plant species without causing symptoms and can effectively induce stable virus-induced gene silencing in plants. Here, we show that pre-infection of ALSV vectors harboring part of a target viral genome (we called ALSV vector vaccines here) inhibits the multiplication and spread of the corresponding challenge viruses [Bean yellow mosaic virus, Zucchini yellow mosaic virus (ZYMV), and Cucumber mosaic virus (CMV)] by a homology-dependent resistance. Further, the plants pre-infected with an ALSV vector having genome sequences of both ZYMV and CMV were protected against double inoculation of ZYMV and CMV. More interestingly, a curative effect of an ALSV vector vaccine could also be expected in ZYMV-infected cucumber plants, because the symptoms subsided on subsequent inoculation with an ALSV vector vaccine. This may be due to the invasion of ALSV, but not ZYMV, in the shoot apical meristem of cucumber.

  19. Dengue vector management using insecticide treated materials and targeted interventions on productive breeding-sites in Guatemala

    PubMed Central

    2012-01-01

    Background In view of the epidemiological expansion of dengue worldwide and the availability of new tools and strategies particularly for controlling the primary dengue vector Aedes aegypti, an intervention study was set up to test the efficacy, cost and feasibility of a combined approach of insecticide treated materials (ITMs) alone and in combination with appropriate targeted interventions of the most productive vector breeding-sites. Methods The study was conducted as a cluster randomized community trial using “reduction of the vector population” as the main outcome variable. The trial had two arms: 10 intervention clusters (neighborhoods) and 10 control clusters in the town of Poptun Guatemala. Activities included entomological assessments (characteristics of breeding-sites, pupal productivity, Stegomyia indices) at baseline, 6 weeks after the first intervention (coverage of window and exterior doorways made of PermaNet 2.0 netting, factory treated with deltamethrin at 55 mg/m2, and of 200 L drums with similar treated material) and 6 weeks after the second intervention (combination of treated materials and other suitable interventions targeting productive breeding-sites i.e larviciding with Temephos, elimination etc.). The second intervention took place 17 months after the first intervention. The insecticide residual activity and the insecticidal content were also studied at different intervals. Additionally, information about demographic characteristics, cost of the intervention, coverage of houses protected and satisfaction in the population with the interventions was collected. Results At baseline (during the dry season) a variety of productive container types for Aedes pupae were identified: various container types holding >20 L, 200 L drums, washbasins and buckets (producing 83.7% of all pupae). After covering 100% of windows and exterior doorways and a small number of drums (where the commercial cover could be fixed) in 970 study households, tropical

  20. Dengue vector management using insecticide treated materials and targeted interventions on productive breeding-sites in Guatemala.

    PubMed

    Rizzo, Nidia; Gramajo, Rodrigo; Escobar, Maria Cabrera; Arana, Byron; Kroeger, Axel; Manrique-Saide, Pablo; Petzold, Max

    2012-10-30

    In view of the epidemiological expansion of dengue worldwide and the availability of new tools and strategies particularly for controlling the primary dengue vector Aedes aegypti, an intervention study was set up to test the efficacy, cost and feasibility of a combined approach of insecticide treated materials (ITMs) alone and in combination with appropriate targeted interventions of the most productive vector breeding-sites. The study was conducted as a cluster randomized community trial using "reduction of the vector population" as the main outcome variable. The trial had two arms: 10 intervention clusters (neighborhoods) and 10 control clusters in the town of Poptun Guatemala. Activities included entomological assessments (characteristics of breeding-sites, pupal productivity, Stegomyia indices) at baseline, 6 weeks after the first intervention (coverage of window and exterior doorways made of PermaNet 2.0 netting, factory treated with deltamethrin at 55 mg/m2, and of 200 L drums with similar treated material) and 6 weeks after the second intervention (combination of treated materials and other suitable interventions targeting productive breeding-sites i.e larviciding with Temephos, elimination etc.). The second intervention took place 17 months after the first intervention. The insecticide residual activity and the insecticidal content were also studied at different intervals. Additionally, information about demographic characteristics, cost of the intervention, coverage of houses protected and satisfaction in the population with the interventions was collected. At baseline (during the dry season) a variety of productive container types for Aedes pupae were identified: various container types holding >20 L, 200 L drums, washbasins and buckets (producing 83.7% of all pupae). After covering 100% of windows and exterior doorways and a small number of drums (where the commercial cover could be fixed) in 970 study households, tropical rains occurred in the area and

  1. A Genetically Engineered Adenovirus Vector Targeted to CD40 Mediates Transduction of Canine Dendritic Cells and Promotes Antigen-Specific Immune Responses In Vivo

    PubMed Central

    Thacker, Erin E.; Nakayama, Masaharu; Smith, Bruce F.; Bird, R. Curtis; Muminova, Zhanat; Strong, Theresa; Timares, Laura; Korokhov, Nikolay; O'Neill, Ann Marie; de Gruijl, Tanja D.; Glasgow, Joel N.; Tani, Kenzaburo; Curiel, David T.

    2009-01-01

    Targeting viral vectors encoding tumor-associated antigens to dendritic cells (DCs) in vivo is likely to enhance the effectiveness of immunotherapeutic cancer vaccines. We have previously shown that genetic modification of adenovirus (Ad) 5 to incorporate CD40 ligand (CD40L) rather than native fiber allows selective transduction and activation of DCs in vitro. Here, we examine the capacity of this targeted vector to induce immune responses to the tumor antigen CEA in a stringent in vivo canine model. CD40-targeted Ad5 transduced canine DCs via the CD40-CD40L pathway in vitro, and following vaccination of healthy dogs, CD40-targeted Ad5 induced strong anti-CEA cellular and humoral responses. These data validate the canine model for future translational studies and suggest targeting of Ad5 vectors to CD40 for in vivo delivery of tumor antigens to DCs is a feasible approach for successful cancer therapy. PMID:19786146

  2. Correction of a deletion mutant by gene targeting with an adenovirus vector.

    PubMed Central

    Wang, Q; Taylor, M W

    1993-01-01

    The usefulness of adenovirus type 5 as a vector for homologous recombination was examined in CHO cells by using the adenine phosphoribosyltransferase (aprt) gene. Infection of a hemizygous CHO APRT- cell line containing a 3-bp deletion in exon 5 of the aprt gene with a recombinant adenovirus containing the wild-type gene resulted in restoration of the APRT+ phenotype at a frequency of 10(-5) to 10(-6) per infected cell. A relatively high frequency (approximately 6 to 20%) of the transductants appears to result from a homologous recombination event. The mutation on the chromosomal aprt gene is corrected in the homologous recombinants, and APRT expression is restored to a normal hemizygous level. Neither adenovirus nor exogenous promoter sequences are detected in the homologous recombinants. The remaining transductants result from random integration of the aprt gene with the adenovirus sequence. A number of adenovirus vectors containing different promoter sequences linked to the hamster aprt gene were constructed. A possible role for the promoter region in the homologous recombination event was indicated by the lack of homologous recombination in constructs lacking an active promoter. Images PMID:8423811

  3. Split vector systems for ultra-targeted gene delivery: a contrivance to achieve ethical assurance of somatic gene therapy in vivo.

    PubMed

    Tolmachov, Oleg E

    2014-08-01

    Tightly controlled spatial localisation of therapeutic gene delivery is essential to maximize the benefits of somatic gene therapy in vivo and to reduce its undesired effects on the 'bystander' cell populations, most importantly germline cells. Indeed, complete ethical assurance of somatic gene therapy can only be achieved with ultra-targeted gene delivery, which excludes the risk of inadvertent germline gene transfer. Thus, it is desired to supplement existing strategies of physical focusing and biological (cell-specific) targeting of gene delivery with an additional principle for the rigid control over spread of gene transfer within the body. In this paper I advance the concept of 'combinatorial' targeting of therapeutic gene transfer in vivo. I hypothesize that it is possible to engineer complex gene delivery vector systems consisting of several components, each one of them capable of independent spread within the human body but incapable of independent facilitation of gene transfer. As the gene delivery augmented by such split vector systems would be reliant on the simultaneous availability of all the vector system components at a predetermined body site, it is envisaged that higher order reaction kinetics required for the assembly of the functional gene transfer configuration would sharpen spatial localisation of gene transfer via curtailing the blurring effect of the vector spread within the body. A particular implementation of such split vector system could be obtained through supplementing a viral therapeutic gene vector with a separate auxiliary vector carrying a non-integrative and non-replicative form of a gene (e.g., mRNA) coding for a cellular receptor of the therapeutic vector component. Gene-transfer-enabling components of the vector system, which would be delivered separately from the vector component loaded with the therapeutic gene cargo, could also be cell-membrane-insertion-proficient receptors, elements of artificial transmembrane channels

  4. Chitosan-graft-polyethylenimine/DNA nanoparticles as novel non-viral gene delivery vectors targeting osteoarthritis.

    PubMed

    Lu, Huading; Dai, Yuhu; Lv, Lulu; Zhao, Huiqing

    2014-01-01

    The development of safe and efficient gene carriers is the key to the clinical success of gene therapy. The present study was designed to develop and evaluate the chitosan-graft-polyethylenimine (CP)/DNA nanoparticles as novel non-viral gene vectors for gene therapy of osteoarthritis. The CP/DNA nanoparticles were produced through a complex coacervation of the cationic polymers with pEGFP after grafting chitosan (CS) with a low molecular weight (Mw) PEI (Mw = 1.8 kDa). Particle size and zeta potential were related to the weight ratio of CP:DNA, where decreases in nanoparticle size and increases in surface charge were observed as CP content increased. The buffering capacity of CP was significantly greater than that of CS. The transfection efficiency of CP/DNA nanoparticles was similar with that of the Lipofectamine™ 2000, and significantly higher than that of CS/DNA and PEI (25 kDa)/DNA nanoparticles. The transfection efficiency of the CP/DNA nanoparticles was dependent on the weight ratio of CP:DNA (w/w). The average cell viability after the treatment with CP/DNA nanoparticles was over 90% in both chondrocytes and synoviocytes, which was much higher than that of PEI (25 kDa)/DNA nanoparticles. The CP copolymers efficiently carried the pDNA inside chondrocytes and synoviocytes, and the pDNA was detected entering into nucleus. These results suggest that CP/DNA nanoparticles with improved transfection efficiency and low cytotoxicity might be a safe and efficient non-viral vector for gene delivery to both chondrocytes and synoviocytes.

  5. A tandem CD19/CD20 CAR lentiviral vector drives on-target and off-target antigen modulation in leukemia cell lines.

    PubMed

    Schneider, Dina; Xiong, Ying; Wu, Darong; Nӧlle, Volker; Schmitz, Sarah; Haso, Waleed; Kaiser, Andrew; Dropulic, Boro; Orentas, Rimas J

    2017-01-01

    Clinical success with chimeric antigen receptor (CAR)- based immunotherapy for leukemia has been accompanied by the associated finding that antigen-escape variants of the disease are responsible for relapse. To target hematologic malignancies with a chimeric antigen receptor (CAR) that targets two antigens with a single vector, and thus potentially lessen the chance of leukemic escape mutations, a tandem-CAR approach was investigated. Antigen binding domains from the FMC63 (anti-CD19) and Leu16 (anti-CD20) antibodies were linked in differing configurations to transmembrane and T cell signaling domains to create tandem-CARs. Expression on the surface of primary human T cells was induced by transduction with a single lentiviral vector (LV) encoding the tandem-CAR. Tandem-CARs were compared to single antigen targeting CARs in vitro and in vivo, and to an admixture of transduced cells expressing each CAR in vivo in immunodeficient (NSG) disease-bearing mice. Tandem constructs efficient killed the Raji leukemia cell line both in vitro and in vivo. Tandem CARs generated less cytokine than the CD20 CAR, but similar to CD19 CARs, on their own. In co-culture experiments at low effector to target ratios with both single- and tandem- CAR-T cells, a rapid down-modulation of full-length CD19 expression was seen on leukemia targets. There also was a partial down-modulation of CD22, and to a lesser degree, of CD20. Our data also highlight the extreme sensitivity of the NALM-6 cell line to general lymphocyte-mediated cytotoxicity. While single and tandem constructs were effective in vivo in a standard setting, in a high-disease burden setting, the tandem CAR proved both effective and less toxic than an admixture of transduced T cell populations expressing single CARs. Tandem CARs are equally effective in standard disease models to single antigen specificity CARs, and may be both more effective and less toxic in a higher disease burden setting. This may be due to optimized cell

  6. Targeting of adenovirus vectors to the LRP receptor family with the high-affinity ligand RAP via combined genetic and chemical modification of the pIX capsomere.

    PubMed

    Corjon, Stéphanie; Wortmann, Andreas; Engler, Tatjana; van Rooijen, Nico; Kochanek, Stefan; Kreppel, Florian

    2008-11-01

    Adenovirus (Ad) vector targeting requires presentation of specific ligands on the virion's surface. Geneti-chemical targeting is based on the genetic introduction of cysteine residues bearing reactive thiol groups into solvent-accessible capsomeres of the virion and subsequent chemical coupling of ligands. Here, we exploited this technology to modify the pIX capsomere with high-affinity ligands. Genetic introduction of C-terminal cysteines to pIX allowed for specific coupling of full-length proteins to the virion, while not affecting vector production. Direct comparison of the two high-affinity ligands receptor- associated protein (RAP) and transferrin (Tf) revealed that targeting after coupling of a high-affinity ligand to pIX presumably requires release of the ligand from its receptor after cell entry. In addition, data obtained by live cell imaging of labeled vector particles demonstrated that coupling of very large proteins to pIX can impair intracellular vector particle trafficking. Finally, we demonstrate that the geneti-chemical targeting technology is suitable for in vivo targeting to liver after intravenous injection. Our data provide significant insight into basic requirements for successful targeting of pIX-modified Ad vectors.

  7. [MicroRNA Target Prediction Based on Support Vector Machine Ensemble Classification Algorithm of Under-sampling Technique].

    PubMed

    Chen, Zhiru; Hong, Wenxue

    2016-02-01

    Considering the low accuracy of prediction in the positive samples and poor overall classification effects caused by unbalanced sample data of MicroRNA (miRNA) target, we proposes a support vector machine (SVM)-integration of under-sampling and weight (IUSM) algorithm in this paper, an under-sampling based on the ensemble learning algorithm. The algorithm adopts SVM as learning algorithm and AdaBoost as integration framework, and embeds clustering-based under-sampling into the iterative process, aiming at reducing the degree of unbalanced distribution of positive and negative samples. Meanwhile, in the process of adaptive weight adjustment of the samples, the SVM-IUSM algorithm eliminates the abnormal ones in negative samples with robust sample weights smoothing mechanism so as to avoid over-learning. Finally, the prediction of miRNA target integrated classifier is achieved with the combination of multiple weak classifiers through the voting mechanism. The experiment revealed that the SVM-IUSW, compared with other algorithms on unbalanced dataset collection, could not only improve the accuracy of positive targets and the overall effect of classification, but also enhance the generalization ability of miRNA target classifier.

  8. Stereotaxic microinjection of viral vectors expressing Cre recombinase to study the role of target genes in cocaine conditioned place preference.

    PubMed

    Schierberl, Kathryn C; Rajadhyaksha, Anjali M

    2013-07-30

    Microinjecting recombinant adenoassociated viral (rAAV) vectors expressing Cre recombinase into distinct mouse brain regions to selectively knockout genes of interest allows for enhanced temporally- and regionally-specific control of gene deletion, compared to existing methods. While conditional deletion can also be achieved by mating mice that express Cre recombinase under the control of specific gene promoters with mice carrying a floxed gene, stereotaxic microinjection allows for targeting of discrete brain areas at experimenter-determined time points of interest. In the context of cocaine conditioned place preference, and other cocaine behavioral paradigms such as self-administration or psychomotor sensitization that can involve withdrawal, extinction and/or reinstatement phases, this technique is particularly useful in exploring the unique contribution of target genes to these distinct phases of behavioral models of cocaine-induced plasticity. Specifically, this technique allows for selective ablation of target genes during discrete phases of a behavior to test their contribution to the behavior across time. Ultimately, this understanding allows for more targeted therapeutics that are best able to address the most potent risk factors that present themselves during each phase of addictive behavior.

  9. Targeting the Immune System to Fight Cancer Using Chemical Receptor Homing Vectors Carrying Polyinosine/Cytosine (PolyIC).

    PubMed

    Levitzki, Alexander

    2012-01-01

    Cancer researchers have been looking for ways to harness the immune system and to reinstate immune surveillance, to kill cancer cells without collateral damage. Here we scan current approaches to targeting the immune system against cancer, and emphasize our own approach. We are using chemical vectors attached to a specific ligand, to introduce synthetic dsRNA, polyinosine/cytosine (polyIC), into tumors. The ligand binds to a receptor protein that is overexpressed on the surface of the tumor cells. Upon ligand binding, the receptor complex is internalized, introducing the polyIC into the cell. In this fashion a large amount of synthetic dsRNA can be internalized, leading to the activation of dsRNA-binding proteins, such as dsRNA dependent protein kinase (PKR), Toll-like receptor 3 (TLR3), retinoic acid-inducible gene I (RIG-1), and melanoma differentiation-associated gene 5 (MDA5). The simultaneous activation of these signaling proteins leads to the rapid demise of the targeted cell and to cytokine secretion. The cytokines lead to a strong bystander effect and to the recruitment of immune cells that converge upon the targeted cells. The bystander effects lead to the destruction of neighboring tumor cells not targeted themselves by the vector. Normal cells, being more robust than tumor cells, survive. This strategy has several advantages: (1) recruitment of the immune system is localized to the tumor. (2) The response is rapid, leading to fast tumor eradication. (3) The bystander effects lead to the eradication of tumor cells not harboring the target. (4) The multiplicity of pro-death signaling pathways elicited by PolyIC minimizes the likelihood of the emergence of resistance. In this chapter we focus on EGFR as the targeted receptor, which is overexpressed in many tumors. In principle, the strategy can be extended to other tumors that overexpress a protein that can be internalized by a ligand, which can be a small molecule, a single chain antibody, or an affibody.

  10. Regulatory Elements in Vectors for Efficient Generation of Cell Lines Producing Target Proteins

    PubMed Central

    Maksimenko, O.; Gasanov, N. B.; Georgiev, P.

    2015-01-01

    To date, there has been an increasing number of drugs produced in mammalian cell cultures. In order to enhance the expression level and stability of target recombinant proteins in cell cultures, various regulatory elements with poorly studied mechanisms of action are used. In this review, we summarize and discuss the potential mechanisms of action of such regulatory elements. PMID:26483956

  11. RNA nanoparticle as a vector for targeted siRNA delivery into glioblastoma mouse model

    PubMed Central

    Lee, Tae Jin; Haque, Farzin; Shu, Dan; Yoo, Ji Young; Li, Hui; Yokel, Robert A.; Horbinski, Craig; Kim, Tae Hyong; Kim, Sung-Hak; Kwon, Chang-Hyuk; Nakano, Ichiro; Kaur, Balveen; Guo, Peixuan; Croce, Carlo M.

    2015-01-01

    Systemic siRNA administration to target and treat glioblastoma, one of the most deadly cancers, requires robust and efficient delivery platform without immunogenicity. Here we report newly emerged multivalent naked RNA nanoparticle (RNP) based on pRNA 3-way-junction (3WJ) from bacteriophage phi29 to target glioblastoma cells with folate (FA) ligand and deliver siRNA for gene silencing. Systemically injected FA-pRNA-3WJ RNPs successfully targeted and delivered siRNA into brain tumor cells in mice, and efficiently reduced luciferase reporter gene expression (4-fold lower than control). The FA-pRNA-3WJ RNP also can target human patient-derived glioblastoma stem cells, thought to be responsible for tumor initiation and deadly recurrence, without accumulation in adjacent normal brain cells, nor other major internal organs. This study provides possible application of pRNA-3WJ RNP for specific delivery of therapeutics such as siRNA, microRNA and/or chemotherapeutic drugs into glioblastoma cells without inflicting collateral damage to healthy tissues. PMID:25885522

  12. RNA nanoparticle as a vector for targeted siRNA delivery into glioblastoma mouse model.

    PubMed

    Lee, Tae Jin; Haque, Farzin; Shu, Dan; Yoo, Ji Young; Li, Hui; Yokel, Robert A; Horbinski, Craig; Kim, Tae Hyong; Kim, Sung-Hak; Kwon, Chang-Hyuk; Nakano, Ichiro; Kaur, Balveen; Guo, Peixuan; Croce, Carlo M

    2015-06-20

    Systemic siRNA administration to target and treat glioblastoma, one of the most deadly cancers, requires robust and efficient delivery platform without immunogenicity. Here we report newly emerged multivalent naked RNA nanoparticle (RNP) based on pRNA 3-way-junction (3WJ) from bacteriophage phi29 to target glioblastoma cells with folate (FA) ligand and deliver siRNA for gene silencing. Systemically injected FA-pRNA-3WJ RNPs successfully targeted and delivered siRNA into brain tumor cells in mice, and efficiently reduced luciferase reporter gene expression (4-fold lower than control). The FA-pRNA-3WJ RNP also can target human patient-derived glioblastoma stem cells, thought to be responsible for tumor initiation and deadly recurrence, without accumulation in adjacent normal brain cells, nor other major internal organs. This study provides possible application of pRNA-3WJ RNP for specific delivery of therapeutics such as siRNA, microRNA and/or chemotherapeutic drugs into glioblastoma cells without inflicting collateral damage to healthy tissues.

  13. Dengue vector dynamics (Aedes aegypti) influenced by climate and social factors in Ecuador: implications for targeted control.

    PubMed

    Stewart Ibarra, Anna M; Ryan, Sadie J; Beltrán, Efrain; Mejía, Raúl; Silva, Mercy; Muñoz, Angel

    2013-01-01

    Dengue fever, a mosquito-borne viral disease, is now the fastest spreading tropical disease globally. Previous studies indicate that climate and human behavior interact to influence dengue virus and vector (Aedes aegypti) population dynamics; however, the relative effects of these variables depends on local ecology and social context. We investigated the roles of climate and socio-ecological factors on Ae. aegypti population dynamics in Machala, a city in southern coastal Ecuador where dengue is hyper-endemic. We studied two proximate urban localities where we monitored weekly Ae. aegypti oviposition activity (Nov. 2010-June 2011), conducted seasonal pupal surveys, and surveyed household to identify dengue risk factors. The results of this study provide evidence that Ae. aegypti population dynamics are influenced by social risk factors that vary by season and lagged climate variables that vary by locality. Best-fit models to predict the presence of Ae. aegypti pupae included parameters for household water storage practices, access to piped water, the number of households per property, condition of the house and patio, and knowledge and perceptions of dengue. Rainfall and minimum temperature were significant predictors of oviposition activity, although the effect of rainfall varied by locality due to differences in types of water storage containers. These results indicate the potential to reduce the burden of dengue in this region by conducting focused vector control interventions that target high-risk households and containers in each season and by developing predictive models using climate and non-climate information. These findings provide the region's public health sector with key information for conducting time and location-specific vector control campaigns, and highlight the importance of local socio-ecological studies to understand dengue dynamics. See Text S1 for an executive summary in Spanish.

  14. Construction of a targeting adenoviral vector carrying AFP promoter for expressing EGFP gene in AFP producing hepatocarcinoma cell

    PubMed Central

    Shi, Yu-Jun; Gong, Jian-Ping; Liu, Chang-An; Li, Xu-Hong; Mei, Ying; Mi, Can; Huo, Yan-Ying

    2004-01-01

    AIM: To construct a recombinant adenoviral vector carrying AFP promoter and EGFP gene for specific expression of EGFP gene in AFP producing hepatocellular carcinoma (HCC) HepG2 cells. METHODS: Based on the Adeno-XTM expression system, the human immediate early cytomegalovirus promoter (PCMV IE) was removed from the plasmid, pshuttle, and replaced by a 0.3 kb α-fetoprotein (AFP) promoter that was synthesized by polymerase chain reaction (PCR). The enhanced green fluorescent protein (EGFP) gene was inserted into the multi-clone site (MCS), and then the recombinant adenovirus vector carrying the 0.3 kb AFP promoter and EGFP gene was constructed. Cells of a normal liver cell line (LO2), a hepatocarcinoma cell line (HepG2) and a cervical cancer cell line (HeLa) were transfected with the adenovirus. Northern blot and fluorescence microscopy were used to detect the expression of the EGFP gene at mRNA or protein level in three different cell lines. RESULTS: The 0.3 kb AFP promoter was synthesized through PCR from the human genome. The AFP promoter and EGFP gene were directly inserted into the plasmid pshuttle as confirmed by restriction digestion and DNA sequencing. Northern blot showed that EGFP gene was markedly transcribed in HepG2 cells, but only slightly in LO2 and HeLa cells. In addition, strong green fluorescence was observed in HepG2 cells under a fluorescence microscopy, but fluorescence was very weak LO2 and HeLa cells. CONCLUSION: Under control of the 0.3 kb human AFP promoter, the recombinant adenovirus vector carrying EGFP gene can be specially expressed in AFP-producing HepG2 cells. Therefore, this adenovirus system can be used as a novel, potent and specific tool for gene-targeting therapy for the AFP positive primary hepatocellular carcinoma. PMID:14716819

  15. CREDVW-Linked Polymeric Micelles As a Targeting Gene Transfer Vector for Selective Transfection and Proliferation of Endothelial Cells.

    PubMed

    Hao, Xuefang; Li, Qian; Lv, Juan; Yu, Li; Ren, Xiangkui; Zhang, Li; Feng, Yakai; Zhang, Wencheng

    2015-06-10

    Nowadays, gene transfer technology has been widely used to promote endothelialization of artificial vascular grafts. However, the lack of gene vectors with low cytotoxicity and targeting function still remains a pressing challenge. Herein, polyethylenimine (PEI, 1.8 kDa or 10 kDa) was conjugated to an amphiphilic and biodegradable diblock copolymer poly(ethylene glycol)-b-poly(lactide-co-glycolide) (mPEG-b-PLGA) to prepare mPEG-b-PLGA-g-PEI copolymers with the aim to develop gene vectors with low cytotoxicity while high transfection efficiency. The micelles were prepared from mPEG-b-PLGA-g-PEI copolymers by self-assembly method. Furthermore, Cys-Arg-Glu-Asp-Val-Trp (CREDVW) peptide was linked to micelle surface to enable the micelles with special recognition for endothelial cells (ECs). In addition, pEGFP-ZNF580 plasmids were condensed into these CREDVW-linked micelles to enhance the proliferation of ECs. These CREDVW-linked micelle/pEGFP-ZNF580 complexes exhibited low cytotoxicity by MTT assay. The cell transfection results demonstrated that pEGFP-ZNF580 could be transferred into ECs efficiently by these micelles. The results of Western blot analysis showed that the relative ZNF580 protein level in transfected ECs increased to 76.9%. The rapid migration of transfected ECs can be verified by wound healing assay. These results indicated that CREDVW-linked micelles could be a suitable gene transfer vector with low cytotoxicity and high transfection efficiency, which has great potential for rapid endothelialization of artificial blood vessels.

  16. Dengue Vector Dynamics (Aedes aegypti) Influenced by Climate and Social Factors in Ecuador: Implications for Targeted Control

    PubMed Central

    Stewart Ibarra, Anna M.; Ryan, Sadie J.; Beltrán, Efrain; Mejía, Raúl; Silva, Mercy; Muñoz, Ángel

    2013-01-01

    Background Dengue fever, a mosquito-borne viral disease, is now the fastest spreading tropical disease globally. Previous studies indicate that climate and human behavior interact to influence dengue virus and vector (Aedes aegypti) population dynamics; however, the relative effects of these variables depends on local ecology and social context. We investigated the roles of climate and socio-ecological factors on Ae. aegypti population dynamics in Machala, a city in southern coastal Ecuador where dengue is hyper-endemic. Methods/Principal findings We studied two proximate urban localities where we monitored weekly Ae. aegypti oviposition activity (Nov. 2010-June 2011), conducted seasonal pupal surveys, and surveyed household to identify dengue risk factors. The results of this study provide evidence that Ae. aegypti population dynamics are influenced by social risk factors that vary by season and lagged climate variables that vary by locality. Best-fit models to predict the presence of Ae. aegypti pupae included parameters for household water storage practices, access to piped water, the number of households per property, condition of the house and patio, and knowledge and perceptions of dengue. Rainfall and minimum temperature were significant predictors of oviposition activity, although the effect of rainfall varied by locality due to differences in types of water storage containers. Conclusions These results indicate the potential to reduce the burden of dengue in this region by conducting focused vector control interventions that target high-risk households and containers in each season and by developing predictive models using climate and non-climate information. These findings provide the region's public health sector with key information for conducting time and location-specific vector control campaigns, and highlight the importance of local socio-ecological studies to understand dengue dynamics. See Text S1 for an executive summary in Spanish. PMID:24324542

  17. Adenoviral vectors coated with PAMAM dendrimer conjugates allow CAR independent virus uptake and targeting to the EGF receptor.

    PubMed

    Vetter, Alexandra; Virdi, Kulpreet S; Espenlaub, Sigrid; Rödl, Wolfgang; Wagner, Ernst; Holm, Per S; Scheu, Christina; Kreppel, Florian; Spitzweg, Christine; Ogris, Manfred

    2013-02-04

    Adenovirus type 5 (Ad) is an efficient gene vector with high gene transduction potential, but its efficiency depends on its native cell receptors coxsackie- and adenovirus receptor (CAR) for cell attachment and α(v)β(3/5) integrins for internalization. To enable transduction of CAR negative cancer cell lines, we have coated the negatively charged Ad by noncovalent charge interaction with cationic PAMAM (polyamidoamine) dendrimers. The specificity for tumor cell infection was increased by targeting the coated Ad to the epidermal growth factor receptor using the peptide ligand GE11, which was coupled to the PAMAM dendrimer via a 2 kDa PEG spacer. Particles were examined by measuring surface charge and size, the degree of coating was determined by transmission electron microscopy. The net positive charge of PAMAM coated Ad enhanced cellular binding and uptake leading to increased transduction efficiency, especially in low to medium CAR expressing cancer cell lines using enhanced green fluorescent protein or luciferase as transgene. While PAMAM coated Ad allowed for efficient internalization, coating with linear polyethylenimine induced excessive particle aggregation, elevated cellular toxicity and lowered transduction efficiency. PAMAM coating of Ad enabled successful transduction of cells in vitro even in the presence of neutralizing antibodies. Taken together, this study clearly proves noncovalent, charge-based coating of Ad vectors with ligand-equipped dendrimers as a viable strategy for efficient transduction of cells otherwise refractory to Ad infection.

  18. Dendritic cell-targeted lentiviral vector immunization uses pseudotransduction and DNA-mediated STING and cGAS activation.

    PubMed

    Kim, Jocelyn T; Liu, Yarong; Kulkarni, Rajan P; Lee, Kevin K; Dai, Bingbing; Lovely, Geoffrey; Ouyang, Yong; Wang, Pin; Yang, Lili; Baltimore, David

    2017-07-21

    Dendritic cell (DC) activation and antigen presentation are critical for efficient priming of T cell responses. Here, we study how lentiviral vectors (LVs) deliver antigen and activate DCs to generate T cell immunization in vivo. We report that antigenic proteins delivered in vector particles via pseudotransduction were sufficient to stimulate an antigen-specific immune response. The delivery of the viral genome encoding the antigen increased the magnitude of this response in vivo but was irrelevant in vitro. Activation of DCs by LVs was independent of MyD88, TRIF, and MAVS, ruling out an involvement of Toll-like receptor or RIG-I-like receptor signaling. Cellular DNA packaged in LV preparations induced DC activation by the host STING (stimulator of interferon genes) and cGAS (cyclic guanosine monophosphate-adenosine monophosphate synthase) pathway. Envelope-mediated viral fusion also activated DCs in a phosphoinositide 3-kinase-dependent but STING-independent process. Pseudotransduction, transduction, viral fusion, and delivery of cellular DNA collaborate to make the DC-targeted LV preparation an effective immunogen. Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

  19. Blood feeding patterns of potential arbovirus vectors of the genus Culex targeting ectothermic hosts

    PubMed Central

    Burkett-Cadena, Nathan D.; Graham, Sean P.; Hassan, Hassan K.; Guyer, Craig; Eubanks, Micky D.; Katholi, Charles R.; Unnasch, Thomas R.

    2014-01-01

    Reptiles and amphibians constitute a significant portion of vertebrate biomass in terrestrial ecosystems and may be important arbovirus reservoirs. To investigate mosquito preference for ectothermic hosts, feeding indices were calculated from data collected in Tuskegee National Forest, Alabama, USA. Four mosquito species fed upon ectothermic hosts, with Cx. peccator and Cx. territans feeding primarily upon ectotherms. These two species appeared to target distinct species with little overlap in host choice. Culex peccator was a generalist in its feeding patterns within ectotherms, while Cx. territans appeared to be a more specialized feeder. Six of eleven ectotherm species fed upon by Cx. territans were fed upon more often than predicted based upon abundance. Spring Peepers were highly preferred over other host species by Cx. territans. Blood meals taken from each host species varied temporally, with some hosts being targeted fairly evenly throughout the season and others being fed upon in seasonal peaks. PMID:18981528

  20. Measurement of the vector analyzing power iT/sub 11/ for the. pi. d. -->. 2p reaction with a polarized target

    SciTech Connect

    Smith, G.R.; Bolger, J.; Boschitz, E.; Mathie, E.L.; Proebstle, G.; Meyer, M.; Vogler, F.; Mango, S.

    1982-06-01

    The vector analyzing power iT/sub 11/ has been measured in the ..pi..d..-->..2p reaction using a vector polarized deuteron target. The data consist of an angular distribution of iT/sub 11/ at T/sub ..pi../ = 256 MeV, and a four point excitation function of iT/sub 11/ taken at a center of mass angle of 55/sup 0/. The angular distribution has been fit with associated Legendre polynomials.

  1. Comparative assessment of diverse strategies for malaria vector population control based on measured rates at which mosquitoes utilize targeted resource subsets.

    PubMed

    Killeen, Gerry F; Kiware, Samson S; Seyoum, Aklilu; Gimnig, John E; Corliss, George F; Stevenson, Jennifer; Drakeley, Christopher J; Chitnis, Nakul

    2014-08-28

    Eliminating malaria requires vector control interventions that dramatically reduce adult mosquito population densities and survival rates. Indoor applications of insecticidal nets and sprays are effective against an important minority of mosquito species that rely heavily upon human blood and habitations for survival. However, complementary approaches are needed to tackle a broader diversity of less human-specialized vectors by killing them at other resource targets. Impacts of strategies that target insecticides to humans or animals can be rationalized in terms of biological coverage of blood resources, quantified as proportional coverage of all blood resources mosquito vectors utilize. Here, this concept is adapted to enable impact prediction for diverse vector control strategies based on measurements of utilization rates for any definable, targetable resource subset, even if that overall resource is not quantifiable. The usefulness of this approach is illustrated by deriving utilization rate estimates for various blood, resting site, and sugar resource subsets from existing entomological survey data. Reported impacts of insecticidal nets upon human-feeding vectors, and insecticide-treated livestock upon animal-feeding vectors, are approximately consistent with model predictions based on measured utilization rates for those human and animal blood resource subsets. Utilization rates for artificial sugar baits compare well with blood resources, and are consistent with observed impact when insecticide is added. While existing data was used to indirectly measure utilization rates for a variety of resting site subsets, by comparison with measured rates of blood resource utilization in the same settings, current techniques for capturing resting mosquitoes underestimate this quantity, and reliance upon complex models with numerous input parameters may limit the applicability of this approach. While blood and sugar consumption can be readily quantified using existing

  2. An Acoustic Signal Enhancement Method Based on Independent Vector Analysis for Moving Target Classification in the Wild.

    PubMed

    Zhao, Qin; Guo, Feng; Zu, Xingshui; Chang, Yuchao; Li, Baoqing; Yuan, Xiaobing

    2017-09-28

    In this paper, we study how to improve the performance of moving target classification by using an acoustic signal enhancement method based on independent vector analysis (IVA) in the unattended ground sensor (UGS) system. Inspired by the IVA algorithm, we propose an improved IVA method based on a microphone array for acoustic signal enhancement in the wild, which adopts a particular multivariate generalized Gaussian distribution as the source prior, an adaptive variable step strategy for the learning algorithm and discrete cosine transform (DCT) to convert the time domain observed signals to the frequency domain. We term the proposed method as DCT-G-IVA. Moreover, we design a target classification system using the improved IVA method for signal enhancement in the UGS system. Different experiments are conducted to evaluate the proposed method for acoustic signal enhancement by comparing with the baseline methods in our classification system under different wild environments. The experimental results validate the superiority of the DCT-G-IVA enhancement method in the classification system for moving targets in the presence of dynamic wind noise.

  3. Classification of a target analyte in solid mixtures using principal component analysis, support vector machines, and Raman spectroscopy

    NASA Astrophysics Data System (ADS)

    O'Connell, Marie-Louise; Howley, Tom; Ryder, Alan G.; Leger, Marc N.; Madden, Michael G.

    2005-06-01

    The quantitative analysis of illicit materials using Raman spectroscopy is of widespread interest for law enforcement and healthcare applications. One of the difficulties faced when analysing illicit mixtures is the fact that the narcotic can be mixed with many different cutting agents. This obviously complicates the development of quantitative analytical methods. In this work we demonstrate some preliminary efforts to try and account for the wide variety of potential cutting agents, by discrimination between the target substance and a wide range of excipients. Near-infrared Raman spectroscopy (785 nm excitation) was employed to analyse 217 samples, a number of them consisting of a target analyte (acetaminophen) mixed with excipients of different concentrations by weight. The excipients used were sugars (maltose, glucose, lactose, sorbitol), inorganic materials (talcum powder, sodium bicarbonate, magnesium sulphate), and food products (caffeine, flour). The spectral data collected was subjected to a number of pre-treatment statistical methods including first derivative and normalisation transformations, to make the data more suitable for analysis. Various methods were then used to discriminate the target analytes, these included Principal Component Analysis (PCA), Principal Component Regression (PCR) and Support Vector Machines.

  4. CD30 Receptor-Targeted Lentiviral Vectors for Human Induced Pluripotent Stem Cell-Specific Gene Modification.

    PubMed

    Friedel, Thorsten; Jung-Klawitter, Sabine; Sebe, Attila; Schenk, Franziska; Modlich, Ute; Ivics, Zoltán; Schumann, Gerald G; Buchholz, Christian J; Schneider, Irene C

    2016-05-01

    Cultures of induced pluripotent stem cells (iPSCs) often contain cells of varying grades of pluripotency. We present novel lentiviral vectors targeted to the surface receptor CD30 (CD30-LV) to transfer genes into iPSCs that are truly pluripotent as demonstrated by marker gene expression. We demonstrate that CD30 expression is restricted to SSEA4(high) cells of human iPSC cultures and a human embryonic stem cell line. When CD30-LV was added to iPSCs during routine cultivation, efficient and exclusive transduction of cells positive for the pluripotency marker Oct-4 was achieved, while retaining their pluripotency. When added during the reprogramming process, CD30-LV solely transduced cells that became fully reprogrammed iPSCs as confirmed by co-expression of endogenous Nanog and the reporter gene. Thus, CD30-LV may serve as novel tool for the selective gene transfer into PSCs with broad applications in basic and therapeutic research.

  5. Intraosseous delivery of lentiviral vectors targeting factor VIII expression in platelets corrects murine hemophilia A.

    PubMed

    Wang, Xuefeng; Shin, Simon C; Chiang, Andy F J; Khan, Iram; Pan, Dao; Rawlings, David J; Miao, Carol H

    2015-04-01

    Intraosseous (IO) infusion of lentiviral vectors (LVs) for in situ gene transfer into bone marrow may avoid specific challenges posed by ex vivo gene delivery, including, in particular, the requirement of preconditioning. We utilized IO delivery of LVs encoding a GFP or factor VIII (FVIII) transgene directed by ubiquitous promoters (a MND or EF-1α-short element; M-GFP-LV, E-F8-LV) or a platelet-specific, glycoprotein-1bα promoter (G-GFP-LV, G-F8-LV). A single IO infusion of M-GFP-LV or G-GFP-LV achieved long-term and efficient GFP expression in Lineage(-)Sca1(+)c-Kit(+) hematopoietic stem cells and platelets, respectively. While E-F8-LV produced initially high-level FVIII expression, robust anti-FVIII immune responses eliminated functional FVIII in circulation. In contrast, IO delivery of G-F8-LV achieved long-term platelet-specific expression of FVIII, resulting in partial correction of hemophilia A. Furthermore, similar clinical benefit with G-F8-LV was achieved in animals with pre-existing anti-FVIII inhibitors. These findings further support platelets as an ideal FVIII delivery vehicle, as FVIII, stored in α-granules, is protected from neutralizing antibodies and, during bleeding, activated platelets locally excrete FVIII to promote clot formation. Overall, a single IO infusion of G-F8-LV was sufficient to correct hemophilia phenotype for long term, indicating that this approach may provide an effective means to permanently treat FVIII deficiency.

  6. [Construction of a recombined adenovirus vector carrying pri-miR-21 gene and research on it's target gene TLR4].

    PubMed

    Zhao, Jing; Xu, Guang-xian; Jia, Wei; Dong, Hui; Zhang, Yi-lin; Zhao, Zhi-jun; Wei, Jun

    2012-02-01

    To construct the recombined adenovirus vector carrying pri-miR-21 gene, which can express mature miR-21 efficiently, and to study the interaction of miR- 21 with its target gene TLR4. Using healthy mouse's gDNA as template, the primary miR-21 coding sequence was amplified by PCR and cloned into a shuttle vector pAdTrack-CMV. Constructed plasmid was sequenced and linearized for homologous recombination with pAdEasy-1 vector in BJ5183 bacteria. The recombined adenovirus vector carrying pri-miR-21 gene was used to challenge HeLa cell. The candidate target gene of miR-21 was determined by miRNA analysis databases. The expression level of TLR4 protein was detected by western blotting. Through the PCR, restriction enzyme digestion, DNA sequencing and expression of GFP, recombinant adenoviral vector pri-miR-21 gene was constructed successfully. Bioinformatic analysis suggested a few possible binding sites between miR-21 and TLR4. Results showed that miR-21 down-regulated TLR4 at protein levels. The recombinant adenoviral vector containing pri- miR-21 was successfully constructed. miR-21 gene interfered with the expression of TLR4 target gene.

  7. Integration profile of retroviral vector in gene therapy treated patients is cell-specific according to gene expression and chromatin conformation of target cell.

    PubMed

    Biasco, Luca; Ambrosi, Alessandro; Pellin, Danilo; Bartholomae, Cynthia; Brigida, Immacolata; Roncarolo, Maria Grazia; Di Serio, Clelia; von Kalle, Christof; Schmidt, Manfred; Aiuti, Alessandro

    2011-02-01

    The analysis of genomic distribution of retroviral vectors is a powerful tool to monitor 'vector-on-host' effects in gene therapy (GT) trials but also provides crucial information about 'host-on-vector' influences based on the target cell genetic and epigenetic state. We had the unique occasion to compare the insertional profile of the same therapeutic moloney murine leukemia virus (MLV) vector in the context of the adenosine deaminase-severe combined immunodeficiency (ADA-SCID) genetic background in two GT trials based on infusions of transduced mature lymphocytes (peripheral blood lymphocytes, PBL) or a single infusion of haematopoietic stem/progenitor cells (HSC). We found that vector insertions are cell-specific according to the differential expression profile of target cells, favouring, in PBL-GT, genes involved in immune system and T-cell functions/pathways as well as T-cell DNase hypersensitive sites, differently from HSC-GT. Chromatin conformations and histone modifications influenced integration preferences but we discovered that only H3K27me3 was cell-specifically disfavoured, thus representing a key epigenetic determinant of cell-type dependent insertion distribution. Our study shows that MLV vector insertional profile is cell-specific according to the genetic/chromatin state of the target cell both in vitro and in vivo in patients several years after GT.

  8. Therapeutic potentialities of EWS-Fli-1 mRNA-targeted vectorized antisense oligonucleotides.

    PubMed

    Maksimenko, A; Lambert, G; Bertrand, J R; Fattal, E; Couvreur, P; Malvy, C

    2003-12-01

    We have used structured antisense oligonucleotides (AON), which are protected against extra and intracellular degradation by their internal structure. We have shown that if correctly designed this structure does not prevent them from hybridizing to the mRNA target. This concept allows reducing the number of thioate groups in the oligonucleotide and therefore the potential toxicity. Junction oncogenes are found in cancers such as certain leukemias, Ewing sarcoma, and thyroid papillary carcinomas. Ewing sarcoma is a cancer of children and young adults with bone metastasis. It is caused by a chromosomic translocation t(11;22) (q24;q12) creating a fusion gene between the genes EWS and Fli-1 giving rise to a chimeric protein which is an unnatural transcription factor. Immortalized NIH/3T3 cells transfected by the EWS-Fli-1 cDNA under the control of the LTR retroviral promoter--which do not undergo apoptosis and which became tumoral--were used for this study. As a model of Ewing sarcoma in nude mice, we have used permanently expressing human EWS-Fli-1 cells grafted to nude mice. The nanospheres or nanocapsules have been used to deliver two different AON: a phosphorothioate, and a structured chimeric AON, both targeted toward the junction area of EWS-Fli-1. Both types of AON-loaded nanoparticles inhibited the growth of the xenografted tumor after intratumoral injections into nude mice, whereas similar nanoparticles with control oligonucleotides had no effect. With AON in nanospheres, we have shown after 24 hours that the mRNA of EWS-Fli-1 was specifically down-regulated, confirming the antisense activity of the targeted AON.

  9. Immunization of Mice with Lentiviral Vectors Targeted to MHC Class II+ Cells Is Due to Preferential Transduction of Dendritic Cells In Vivo

    PubMed Central

    Ciré, Séverine; Da Rocha, Sylvie; Yao, Roseline; Fisson, Sylvain; Buchholz, Christian J.; Collins, Mary K.; Galy, Anne

    2014-01-01

    Gene transfer vectors such as lentiviral vectors offer versatile possibilities to express transgenic antigens for vaccination purposes. However, viral vaccines leading to broad transduction and transgene expression in vivo, are undesirable. Therefore, strategies capable of directing gene transfer only to professional antigen-presenting cells would increase the specific activity and safety of genetic vaccines. A lentiviral vector pseudotype specific for murine major histocompatibilty complex class II (LV-MHCII) was recently developed and the present study aims to characterize the in vivo biodistribution profile and immunization potential of this vector in mice. Whereas the systemic administration of a vector pseudotyped with a ubiquitously-interacting envelope led to prominent detection of vector copies in the liver of animals, the injection of an equivalent amount of LV-MHCII resulted in a more specific biodistribution of vector and transgene. Copies of LV-MHCII were found only in secondary lymphoid organs, essentially in CD11c+ dendritic cells expressing the transgene whereas B cells were not efficiently targeted in vivo, contrary to expectations based on in vitro testing. Upon a single injection of LV-MHCII, naive mice mounted specific effector CD4 and CD8 T cell responses against the intracelllular transgene product with the generation of Th1 cytokines, development of in vivo cytotoxic activity and establishment of T cell immune memory. The targeting of dendritic cells by recombinant viral vaccines must therefore be assessed in vivo but this strategy is feasible, effective for immunization and cross-presentation and constitutes a potentially safe alternative to limit off-target gene expression in gene-based vaccination strategies with integrative vectors. PMID:25058148

  10. Innocuous full-length botulinum neurotoxin targets and promotes the expression of lentiviral vectors in central and autonomic neurons.

    PubMed

    O'Leary, V B; Ovsepian, S V; Raghunath, A; Huo, Q; Lawrence, G W; Smith, L; Dolly, J O

    2011-07-01

    Fragments of botulinum neurotoxin (BoNT) have been explored as potential targeting moieties and carriers of biomolecules into neurons, although with lower binding and translocation efficiency compared with intact proteins. This study exploits a detoxified recombinant form of full-length BoNT/B (BoTIM/B) fused with core streptavidin (CS-BoTIM/B) for lentiviral targeting to central and autonomic neurons. CS-BoTIM/B underwent an activity-dependent entry into cultured spinal cord neurons. Coupling CS-BoTIM/B to biotinylated lentivirus-encoding green fluorescent protein (GFP) endowed considerable neuron selectivity to the vector as evident from the preferential expression of the reporter in neurons co-cultured with skeletal muscle cells. CS-BoTIM/B-guided lentiviral transduction with the expression of a SNARE protein, SNAP-25 (S25), rendered non-susceptible to proteolysis by three BoNT serotypes, yielded a sizable decrease in cleaved S25 upon exposure of spinal cord neurons to these toxins. This was accompanied by synaptic transmission being spared from blockade by BoNT/A or BoNT/E, reflecting adequate translation and functional competence of recombinant multi-toxin-resistant S25. The augmented neurotropism conveyed on the lentivirus by CS-BoTIM/B was also demonstrated in vivo through enhanced expression of a reporter in intramural ganglionic neurons in the rat trachea, after injection of the targeted GFP-encoding lentivirus. Thus, a novel and realistic prospect for gene therapy of peripheral neuropathies is offered in this study through lentiviral targeting to neurons by CS-BoTIM/B.

  11. Broad RTK-targeted therapy overcomes molecular heterogeneity-driven resistance to cetuximab via vectored immunoprophylaxis in colorectal cancer.

    PubMed

    Hu, Shi; Dai, Haibin; Li, Tian; Tang, Ying; Fu, Wenyan; Yuan, Qingning; Wang, Feifei; Lv, Gaojian; Lv, Yuanyuan; Fan, Xiaoyan; Zhang, Sheng; Jin, Ruobing; Shen, Yafeng; Lin, Fangxing; Ye, Xuting; Ding, Min; Yang, Yongji; Lei, Changhai

    2016-11-01

    The human epidermal growth factor receptor (EGFR) targeting chimeric monoclonal antibody, cetuximab (Erbitux®), is a widely used drug in the treatment of metastatic colorectal cancer. However, the activation of the extensive crosstalk among the EGFR family receptors as well as other tyrosine kinase receptors (RTKs) impairs the efficacy of the drug by fueling acquired resistance. To identify the responsible potential activation pathway underlying cetuximab resistance and generate novel treatment strategies, cetuximab-resistant colorectal cancer cell lines were generated and validated and a functional RNAi screen targeting human RTKs was used to identify extensive receptor tyrosine kinase signaling networks established in resistant cancer cells. MET, Axl, and IGF-1R were identified as contributors to the acquired resistance to cetuximab. Targeting vectored immunoprophylaxis (VIPs) to different RTKs were generated and characterized. Different VIP approaches were evaluated in vivo with parental and cetuximab-resistance xenografts and the RTKs in resistant cancer xenografts were inhibited with VIPs via re-sensitization to cetuximab treatment. Combination of VIPs was more broadly efficacious, mechanistically, due to co-blocking the EGFR/Axl/MET signaling pathway, which was cross-activated in the resistant cell lines. Moreover, a VIP-based procedural treatment strategy not only eliminated the tumor but also afforded long-lasting protection from tumor recurrence and resistance. Overall, EGFR-related RTK pathway-network activation represents a novel mechanism underlying cetuximab resistance. A broad VIP combination strategy and VIP-based procedural treatment strategy may be a recommended addition to cetuximab-based targeted therapy. Our results establish a new principle to achieve combined RTK inhibition and reverse drug resistance using a VIP approach.

  12. Evaluation of a combinatorial RNAi lentivirus vector targeting foot-and-mouth disease virus in vitro and in vivo

    PubMed Central

    ZHANG, XIAOXI; ZHENG, HAIXUE; XU, MINJUN; ZHOU, YU; LI, XIANGPING; YANG, FAN; LIU, QINGYOU; SHI, DESHUN

    2015-01-01

    Foot-and-mouth disease virus (FMDV) causes a highly contagious disease of cloven-hoofed animals, which leads to serious economical losses. FMDV is not adequately controlled by vaccination or biosecurity measures. To generate genetically modified FMDV-resistant animals, a combinatorial expression cassette producing three short hairpin (sh) RNAs was constructed using the lentivirus (LV) vector, LV-3shRNA. The three shRNAs were expressed under the regulation of DNA polymerase III promoters from a buffalo and a bovine source, with one targeted to the non-structural protein 3B, and the other two targeted to the viral polymerase protein 3D of FMDV, respectively. The role of LV-3shRNA in the inhibition of the replication of FMDV was determined in BHK-21 cells and in suckling mice. The results revealed that LV-3shRNA reduced viral growth 3-fold (24 h post-infection) when the cells were challenged with 107-times the tissue culture infective dose (TCID50)/ml of O serotype FMDV. The suckling mice pretreated with LV-3shRNA were completely protected on administration of 5-times the dose of FMDV otherwise sufficient to kill 50% of the experimental animals (LD50). These results demonstrated that the LV-mediated dual expression of three FMDV-specific shRNAs provided a novel strategy towards combating FMDV, which facilitates the permanent introduction of novel disease-resistance traits into the buffalo and bovine genomes in the future. PMID:26323462

  13. DENDRITIC CELL SUBSETS AS VECTORS AND TARGETS FOR IMPROVED CANCER THERAPY

    PubMed Central

    Palucka, Karolina; Ueno, Hideki; Roberts, Lee; Fay, Joseph; Banchereau, Jacques

    2010-01-01

    SUMMARY Current active immunotherapy trials have shown durable tumor regressions in a fraction of patients. However, clinical efficacy of current vaccines is limited, possibly because tumors skew the immune system by means of myeloid-derived suppressor cells, inflammatory type 2 T cells and regulatory T cells (Tregs), all of which prevent the generation of effector cells. To improve the clinical efficacy of cancer vaccines in patients with metastatic disease, we need to design novel and improved strategies that can boost adaptive immunity to cancer, help overcome Tregs and allow the breakdown of the immunosuppressive tumor microenvironment. This can be achieved by exploiting the fast increasing knowledge about the dendritic cell (DC) system, including the existence of distinct DC subsets. Critical to the design of better vaccines is the concept of distinct DC subsets and distinct DC activation pathways, all contributing to the generation of unique adaptive immune responses. Such novel DC vaccines will be used as monotherapy in patients with resected disease and in combination with antibodies and/or drugs targeting suppressor pathways and modulation of the tumor environment in patients with metastatic disease. PMID:20490776

  14. Targeted Delivery of Human VEGF Gene via Complexes of Magnetic Nanoparticle-Adenoviral Vectors Enhanced Cardiac Regeneration

    PubMed Central

    Ou, Lailiang; Wang, Weiwei; Delyagina, Evgenya; Lux, Cornelia; Sorg, Heiko; Riehemann, Kristina

    2012-01-01

    This study assessed the concept of whether delivery of magnetic nanobeads (MNBs)/adenoviral vectors (Ad)–encoded hVEGF gene (AdhVEGF) could regenerate ischaemically damaged hearts in a rat acute myocardial infarction model under the control of an external magnetic field. Adenoviral vectors were conjugated to MNBs with the Sulfo-NHS-LC-Biotin linker. In vitro transduction efficacy of MNBs/Ad–encoded luciferase gene (Adluc) was compared with Adluc alone in human umbilical vein endothelial cells (HUVECs) under magnetic field stimulation. In vivo, in a rat acute myocardial infarction (AMI) model, MNBs/AdhVEGF complexes were injected intravenously and an epicardial magnet was employed to attract the circulating MNBs/AdhVEGF complexes. In vitro, compared with Adluc alone, MNBs/Adluc complexes had a 50-fold higher transduction efficiency under the magnetic field. In vivo, epicardial magnet effectively attracted MNBs/AdhVEGF complexes and resulted in strong therapeutic gene expression in the ischemic zone of the infarcted heart. When compared to other MI-treated groups, the MI-M+/AdhVEGF group significantly improved left ventricular function (p<0.05) assessed by pressure-volume loops after 4 weeks. Also the MI-M+/AdhVEGF group exhibited higher capillary and arteriole density and lower collagen deposition than other MI-treated groups (p<0.05). Magnetic targeting enhances transduction efficiency and improves heart function. This novel method to improve gene therapy outcomes in AMI treatment offers the potential into clinical applications. PMID:22844395

  15. An Electron Fixed Target Experiment to Search for a New Vector Boson A' Decaying to e+e-

    SciTech Connect

    Essig, Rouven; Schuster, Philip; Toro, Natalia; Wojtsekhowski, Bogdan; /Jefferson Lab

    2010-06-11

    We describe an experiment to search for a new vector boson A' with weak coupling {alpha}' {approx}> 6 x 10{sup -8} {alpha} to electrons ({alpha} = e{sup 2}/4{pi}) in the mass range 65 MeV < m{sub A'} < 550 MeV. New vector bosons with such small couplings arise naturally from a small kinetic mixing of the 'dark photon' A' with the photon - one of the very few ways in which new forces can couple to the Standard Model - and have received considerable attention as an explanation of various dark matter related anomalies. A' bosons are produced by radiation off an electron beam, and could appear as narrow resonances with small production cross-section in the trident e{sup +}e{sup -} spectrum. We summarize the experimental approach described in a proposal submitted to Jefferson Laboratory's PAC35, PR-10-009. This experiment, the A' Experiment (APEX), uses the electron beam of the Continuous Electron Beam Accelerator Facility at Jefferson Laboratory (CEBAF) at energies of {approx} 1-4 GeV incident on 0.5-10% radiation length Tungsten wire mesh targets, and measures the resulting e{sup +}e{sup -} pairs to search for the A' using the High Resolution Spectrometer and the septum magnet in Hall A. With a {approx} 1 month run, APEX will achieve very good sensitivity because the statistics of e{sup +}e{sup -} pairs will be {approx} 10,000 times larger in the explored mass range than any previous search for the A' boson. These statistics and the excellent mass resolution of the spectrometers allow sensitivity to {alpha}'/{alpha} one to three orders of magnitude below current limits, in a region of parameter space of great theoretical and phenomenological interest. Similar experiments could also be performed at other facilities, such as the Mainz Microtron.

  16. An electron fixed target experiment to search for a new vector boson A' decaying to e+e-

    DOE PAGES

    Rouven Essig; Schuster, Philip; Toro, Natalia; ...

    2011-02-02

    We describe an experiment to search for a new vector boson A' with weak coupling alpha' > 6 x 10–8 α to electrons (α' = e2/4π) in the mass range 65 MeV < mA' < 550 MeV. New vector bosons with such small couplings arise naturally from a small kinetic mixing of the "dark photon" A' with the photon -- one of the very few ways in which new forces can couple to the Standard Model -- and have received considerable attention as an explanation of various dark matter related anomalies. A' bosons are produced by radiation off an electronmore » beam, and could appear as narrow resonances with small production cross-section in the trident e+e- spectrum. We summarize the experimental approach described in a proposal submitted to Jefferson Laboratory's PAC35, PR-10-009. This experiment, the A' Experiment (APEX), uses the electron beam of the Continuous Electron Beam Accelerator Facility at Jefferson Laboratory (CEBAF) at energies of ~1-4 GeV incident on 0.5-10% radiation length Tungsten wire mesh targets, and measures the resulting e+e- pairs to search for the A' using the High Resolution Spectrometer and the septum magnet in Hall A. With a ~1 month run, APEX will achieve very good sensitivity because the statistics of e+e- pairs will be ~10,000 times larger in the explored mass range than any previous search for the A' boson. These statistics and the excellent mass resolution of the spectrometers allow sensitivity to α'/α one to three orders of magnitude below current limits, in a region of parameter space of great theoretical and phenomenological interest. Similar experiments could also be performed at other facilities, such as the Mainz Microtron.« less

  17. Tumor-targeted efficiency of shRNA vector harboring chimera hTERT/U6 promoter.

    PubMed

    Zhang, Penghui; Chen, Yaqin; Jiang, Xixin; Tu, Zhiguang; Zou, Lin

    2010-05-01

    Telomerase is closely related to tumor, and hTERT is the rate-limiting factor for telomerase activity. The transcription and expression of hTERT is determined by hTERT promoter, which has the ability of anchoring telomerase positive cells. RNA interference (RNAi) has been potentially used in the functional genomics and gene therapy recently. However, the limitations of RNAi uncertain interference and safety hamper its wide applications. To overcome these limitations, we constructed shRNA vectors harboring either U6 promoter or chimera hTERT/U6 promoter aiming at EGFP and hTERT genes (shRNA-EGFP-U6, shRNA-EGFP-hTERT/U6, shRNA-hTERT-U6 and shRNA-hTERT-hTERT/U6), to suppress the expression of GFP and hTERT in telomerase negative human normal fibroblast HELF cells and telomerase positive human hepatocarcinoma SMMC-7721 and HepG2 cells, respectively. HELF-EGFP and SMMC-7721-EGFP cells stably expressing EGFP or hTERT were constructed. GFP expression was inhibited in both HELF-EGFP and SMMC-7721-EGFP cells expressing shRNA-EGFP-U6. Further results showed that GFP expression was suppressed only in telomerase positive SMMC-7721 cells and HepG2 cells, but not in telomerase negative HELF cells expressing shRNA-EGFP-hTERT/U6. Further results found that hTERT expression was effectively inhibited from liver cancer cells expressing shRNA-hTERT-U6 or shRNA-hTERT-hTERT/U6 both in vitro and in vivo. Our study illustrates the tumor-targeted efficiency of shRNA vectors harboring chimera hTERT/U6 promoter in telomerase positive cells, which will benefit tumor therapy.

  18. Co-expression of multiple target proteins in plants from a tobacco mosaic virus vector using a combination of homologous and heterologous subgenomic promoters.

    PubMed

    Roy, Gourgopal; Weisburg, Sangeetha; Foy, Kelly; Rabindran, Shailaja; Mett, Vadim; Yusibov, Vidadi

    2011-11-01

    To co-express multiple target proteins, we engineered a single-component chimeric tobacco mosaic virus (TMV)-based vector containing homologous and heterologous capsid protein subgenomic RNA promoters. Delivery of this vector into Nicotiana benthamiana plants via agroinfiltration resulted in co-expression of two reporter genes within a single cell. Furthermore, co-expression of a host-specific antisense RNA or a silencing suppressor protein from this vector augmented the accumulation of green fluorescent protein or a vaccine antigen, hemagglutinin from avian influenza virus A/Vietnam/1194/04. These findings suggest that this chimeric vector utilizing the homologous and heterologous subgenomic TMV promoters has a potential for high-level production of multiple therapeutic proteins including monoclonal antibodies.

  19. Integration profile of retroviral vector in gene therapy treated patients is cell-specific according to gene expression and chromatin conformation of target cell

    PubMed Central

    Biasco, Luca; Ambrosi, Alessandro; Pellin, Danilo; Bartholomae, Cynthia; Brigida, Immacolata; Roncarolo, Maria Grazia; Di Serio, Clelia; von Kalle, Christof; Schmidt, Manfred; Aiuti, Alessandro

    2011-01-01

    The analysis of genomic distribution of retroviral vectors is a powerful tool to monitor ‘vector-on-host’ effects in gene therapy (GT) trials but also provides crucial information about ‘host-on-vector’ influences based on the target cell genetic and epigenetic state. We had the unique occasion to compare the insertional profile of the same therapeutic moloney murine leukemia virus (MLV) vector in the context of the adenosine deaminase-severe combined immunodeficiency (ADA-SCID) genetic background in two GT trials based on infusions of transduced mature lymphocytes (peripheral blood lymphocytes, PBL) or a single infusion of haematopoietic stem/progenitor cells (HSC). We found that vector insertions are cell-specific according to the differential expression profile of target cells, favouring, in PBL-GT, genes involved in immune system and T-cell functions/pathways as well as T-cell DNase hypersensitive sites, differently from HSC-GT. Chromatin conformations and histone modifications influenced integration preferences but we discovered that only H3K27me3 was cell-specifically disfavoured, thus representing a key epigenetic determinant of cell-type dependent insertion distribution. Our study shows that MLV vector insertional profile is cell-specific according to the genetic/chromatin state of the target cell both in vitro and in vivo in patients several years after GT. PMID:21243617

  20. Targeted deletion of the gp72 gene decreases the infectivity of Trypanosoma cruzi for mice and insect vectors.

    PubMed

    Basombrío, Miguel A; Gómez, Laura; Padilla, Angel M; Ciaccio, Mirella; Nozaki, Tomoyoshi; Cross, George A M

    2002-06-01

    The infective behavior of a mutant Trypanosoma cruzi clone, carrying a targeted deletion of the gp72 gene, was studied in the insect vector Triatoma infestans and in mice. After feeding T. infestans with complement-resistant forms (CRF) of Ynull and wild-type clones, it was observed that the number of parasites released in the bug's feces was reduced to less than 1% in the mutant clone. Both gp72-null and wild-type clones had a low infectivity for mice in comparison with other T. cruzi isolates, probably as a consequence of prolonged in vitro culture. Therefore, the behavior of both clones was tested in highly susceptible BALB suckling mice and immunodeficient athymic mice. After infecting the animals with 10(5) CRF, wild-type parasites could be detected in fresh blood mounts of most mice, but mutants were never found by this method. However, in 4 of 22 hemocultures from 11 athymic mice, gp72-null epimastigotes carrying the mutant phenotype were reisolated by day 29 of infection. Serological and polymerase chain reaction determinations performed on the blood of animals inoculated with the mutants indicated the possibility of temporary infections, which were extinguished after 90 days. The intact GP72 gene seems essential for sustaining latent infections in immunocompetent animals.

  1. Chemical composition, toxicity and non-target effects of Pinus kesiya essential oil: An eco-friendly and novel larvicide against malaria, dengue and lymphatic filariasis mosquito vectors.

    PubMed

    Govindarajan, Marimuthu; Rajeswary, Mohan; Benelli, Giovanni

    2016-07-01

    Mosquitoes (Diptera: Culicidae) are vectors of important parasites and pathogens causing death, poverty and social disability worldwide, with special reference to tropical and subtropical countries. The overuse of synthetic insecticides to control mosquito vectors lead to resistance, adverse environmental effects and high operational costs. Therefore, the development of eco-friendly control tools is an important public health challenge. In this study, the mosquito larvicidal activity of Pinus kesiya leaf essential oil (EO) was evaluated against the malaria vector Anopheles stephensi, the dengue vector Aedes aegypti and the lymphatic filariasis vector Culex quinquefasciatus. The chemical composition of the EO was analyzed by gas chromatography-mass spectroscopy. GC-MS revealed that the P. kesiya EO contained 18 compounds. Major constituents were α-pinene, β-pinene, myrcene and germacrene D. In acute toxicity assays, the EO showed significant toxicity against early third-stage larvae of An. stephensi, Ae. aegypti and Cx. quinquefasciatus, with LC50 values of 52, 57, and 62µg/ml, respectively. Notably, the EO was safer towards several aquatic non-target organisms Anisops bouvieri, Diplonychus indicus and Gambusia affinis, with LC50 values ranging from 4135 to 8390µg/ml. Overall, this research adds basic knowledge to develop newer and safer natural larvicides from Pinaceae plants against malaria, dengue and filariasis mosquito vectors.

  2. In vitro incorporation of a cell-binding protein to a lentiviral vector using an engineered split intein enables targeted delivery of genetic cargo.

    PubMed

    Chamoun-Emanuelli, Ana M; Wright, Gus; Roger, Smith; Münch, Robert C; Buchholz, Christian J; Chen, Zhilei

    2015-12-01

    Gene therapy represents a promising therapeutic paradigm for addressing many disorders, but the absence of a vector that can be robustly and reproducibly functionalized with cell-homing functionality to mediate the delivery of genetic cargo specifically to target cells following systemic administration has stood as a major impediment. In this study, a high-affinity protein-protein pair comprising a splicing-deficient naturally split intein was used as molecular Velcro to append a HER2/neu-binding protein (DARPin) onto the surface of a binding-deficient, fusion-competent lentivirus. HER2/neu-specific lentiviruses created using this in vitro pseudotyping approach were able to deliver their genetic reporter cargo specifically to cells that express the target receptor at high levels in a co-culture. We envision that the described technology could provide a powerful, broadly applicable platform for the incorporation of cell-targeting functionality onto viral vectors.

  3. Low-Affinity Neurotrophin Receptor p75 Promotes the Transduction of Targeted Lentiviral Vectors to Cholinergic Neurons of Rat Basal Forebrain.

    PubMed

    Antyborzec, Inga; O'Leary, Valerie B; Dolly, James O; Ovsepian, Saak V

    2016-10-01

    Basal forebrain cholinergic neurons (BFCNs) are one of the most affected neuronal types in Alzheimer's disease (AD), with their extensive loss documented at late stages of the pathology. While discriminatory provision of neuroprotective agents and trophic factors to these cells is thought to be of substantial therapeutic potential, the intricate topography and structure of the forebrain cholinergic system imposes a major challenge. To overcome this, we took advantage of the physiological enrichment of BFCNs with a low-affinity p75 neurotrophin receptor (p75(NTR)) for their targeting by lentiviral vectors within the intact brain of adult rat. Herein, a method is described that affords selective and effective transduction of BFCNs with a green fluorescence protein (GFP) reporter, which combines streptavidin-biotin technology with anti-p75(NTR) antibody-coated lentiviral vectors. Specific GFP expression in cholinergic neurons was attained in the medial septum and nuclei of the diagonal band Broca after a single intraventricular administration of such targeted vectors. Bioelectrical activity of GFP-labeled neurons was proven to be unchanged. Thus, proof of principle is obtained for the utility of the low-affinity p75(NTR) for targeted transduction of vectors to BFCNs in vivo.

  4. Mosquitocidal properties of IgG targeting the glutamate-gated chloride channel in three mosquito disease vectors (Diptera: Culicidae).

    PubMed

    Meyers, Jacob I; Gray, Meg; Foy, Brian D

    2015-05-15

    The glutamate-gated chloride channel (GluCl) is a highly sensitive insecticide target of the avermectin class of insecticides. As an alternative to using chemical insecticides to kill mosquitoes, we tested the effects of purified immunoglobulin G (IgG) targeting the extracellular domain of GluCl from Anopheles gambiae (AgGluCl) on the survivorship of three key mosquito disease vectors: Anopheles gambiae s.s., Aedes aegypti and Culex tarsalis. When administered through a single blood meal, anti-AgGluCl IgG reduced the survivorship of A. gambiae in a dose-dependent manner (LC50: 2.82 mg ml(-1), range 2.68-2.96 mg ml(-1)) but not A. aegypti or C. tarsalis. We previously demonstrated that AgGluCl is only located in tissues of the head and thorax of A. gambiae. To verify that AgGluCl IgG is affecting target antigens found outside the midgut, we injected it directly into the hemocoel via intrathoracic injection. A single, physiologically relevant concentration of anti-AgGluCl IgG injected into the hemocoel equally reduced mosquito survivorship of all three species. To test whether anti-AgGluCl IgG was entering the hemocoel of each of these mosquitoes, we fed mosquitoes a blood meal containing anti-AgGluCl IgG and subsequently extracted their hemolymph. We only detected IgG in the hemolymph of A. gambiae, suggesting that resistance of A. aegypti and C. tarsalis to anti-AgGluCl IgG found in blood meals is due to deficient IgG translocation across the midgut. We predicted that anti-AgGluCl IgG's mode of action is by antagonizing GluCl activity. To test this hypothesis, we fed A. gambiae blood meals containing anti-AgGluCl IgG and the GluCl agonist ivermectin (IVM). Anti-AgGluCl IgG attenuated the mosquitocidal effects of IVM, suggesting that anti-AgGluCl IgG antagonizes IVM-induced activation of GluCl. Lastly, we stained adult, female A. aegypti and C. tarsalis for GluCl expression. Neuronal GluCl expression in these mosquitoes was similar to previously reported A

  5. Assessment of mosquito larval productivity among different land use types for targeted malaria vector control in the western Kenya highlands.

    PubMed

    Kweka, Eliningaya J; Munga, Stephen; Himeidan, Yousif; Githeko, Andrew K; Yan, Guyuin

    2015-07-05

    Mosquito larval source management (LSM) is likely to be more effective when adequate information such as dominant species, seasonal abundance, type of productive habitat, and land use type are available for targeted sites. LSM has been an effective strategy for reducing malaria morbidity in both urban and rural areas in Africa where sufficient proportions of larval habitats can be targeted. In this study, we conducted longitudinal larval source surveillance in the western Kenya highlands, generating data which can be used to establish cost-effective targeted intervention tools. One hundred and twenty-four (124) positive larval habitats were monitored weekly and sampled for mosquito larvae over the 85-week period from 28 July 2009 to 3 March 2011. Two villages in the western Kenya highlands, Mbale and Iguhu, were included in the study. After preliminary sampling, habitats were classified into four types: hoof prints (n = 21; 17 % of total), swamps (n = 32; 26%), abandoned goldmines (n = 35; 28%) and drainage ditches (n = 36; 29%). Positive habitats occurred in two land use types: farmland (66) and pasture (58). No positive larval habitats occurred in shrub land or forest. A total of 46,846 larvae were sampled, of which 44.1% (20,907) were from abandoned goldmines, 30.9% (14,469) from drainage ditches, 22.4% (10,499) from swamps and 2.1% (971) from hoof prints. In terms of land use types, 57.2% (26,799) of the sampled larvae were from pasture and 42.8% (20,047) were from farmland. Of the specimens identified morphologically, 24,583 (52.5%) were Anopheles gambiae s.l., 11,901 (25.4%) were Culex quinquefasciatus, 5628 (12%) were An. funestus s.l. and 4734 (10.1%) were other anopheline species (An. coustani, An. squamosus, An. ziemanni or An. implexus). Malaria vector dynamics varied seasonally, with An.gambiae s.s. dominating during wet season and An.arabiensis during dry season. An increased proportion of An. arabiensis was observed compared to

  6. Construction of interference vector targeting Ep-CAM gene and its effects on colorectal cancer cell proliferation.

    PubMed

    Qi, Yanmei; Zhou, Fengqiang; Zhang, Lu; Liu, Lei; Xu, Hong; Guo, Huiguang

    2015-01-01

    Prior study indicates that abnormal protein expression and functional changes in the development and progression of colorectal cancer is related to gene expression. The aim of this study was to construct an interference plasmid targeting the Ep-CAM gene and to investigate its effects on the proliferation of colorectal cancer cells. In this study, HT-29 and HCT-116 colorectal cancer cell lines were selected as cell models. The double-stranded micro (mi)RNA oligo was inserted into the pcDNATM6.2-GW/EmGFPmiR vector, which is an expression of miRNA. Lipofectamine™ 2000 was used to transfer plasmid into the empty plasmid group (transfected pcDNATM6.2-GW/EmGFPmiR-neg) and the interference group (transfected pcDNATM6.2-GW/EmGFPmiR-Ep-CAM-1), respectively. Meanwhile, the nontransferred HT-29 and HCT-116 acts as the blank control group. Reverse transcription polymerase chain reaction (RT-PCR) was used to detect the transfection efficiency. Western blot was used to detect Ep-CAM protein expression. The cell proliferation in each group was detected by using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The results indicated that the Ep-CAM messenger (m)RNA expression in the interference group was lower significantly compared with that of the empty plasmid group and control group (P<0.01). Western blot analysis results showed that Ep-CAM protein expression was significantly lower in interference group compared with that of the empty plasmid group and the control group (P<0.01). MTT assay results demonstrated that the proliferation ability of cells in the interference group was significantly inhibited compared with the two other groups (P<0.05). Silencing of Ep-CAM can significantly inhibit the proliferation of colorectal cancer cells.

  7. Mechanisms of Pyrethroid Resistance in the Dengue Mosquito Vector, Aedes aegypti: Target Site Insensitivity, Penetration, and Metabolism

    PubMed Central

    Kasai, Shinji; Komagata, Osamu; Itokawa, Kentaro; Shono, Toshio; Ng, Lee Ching; Kobayashi, Mutsuo; Tomita, Takashi

    2014-01-01

    Aedes aegypti is the major vector of yellow and dengue fevers. After 10 generations of adult selection, an A. aegypti strain (SP) developed 1650-fold resistance to permethrin, which is one of the most widely used pyrethroid insecticides for mosquito control. SP larvae also developed 8790-fold resistance following selection of the adults. Prior to the selections, the frequencies of V1016G and F1534C mutations in domains II and III, respectively, of voltage-sensitive sodium channel (Vssc, the target site of pyrethroid insecticide) were 0.44 and 0.56, respectively. In contrast, only G1016 alleles were present after two permethrin selections, indicating that G1016 can more contribute to the insensitivity of Vssc than C1534. In vivo metabolism studies showed that the SP strain excreted permethrin metabolites more rapidly than a susceptible SMK strain. Pretreatment with piperonyl butoxide caused strong inhibition of excretion of permethrin metabolites, suggesting that cytochrome P450 monooxygenases (P450s) play an important role in resistance development. In vitro metabolism studies also indicated an association of P450s with resistance. Microarray analysis showed that multiple P450 genes were over expressed during the larval and adult stages in the SP strain. Following quantitative real time PCR, we focused on two P450 isoforms, CYP9M6 and CYP6BB2. Transcription levels of these P450s were well correlated with the rate of permethrin excretion and they were certainly capable of detoxifying permethrin to 4′-HO-permethrin. Over expression of CYP9M6 was partially due to gene amplification. There was no significant difference in the rate of permethrin reduction from cuticle between SP and SMK strains. PMID:24945250

  8. One-pot fabrication of silver nanocrystals using Nicandra physalodes: A novel route for mosquito vector control with moderate toxicity on non-target water bugs.

    PubMed

    Govindarajan, Marimuthu; Khater, Hanem F; Panneerselvam, Chellasamy; Benelli, Giovanni

    2016-08-01

    Mosquitoes (Diptera: Culicidae) as vectors for important diseases and parasites causing millions of deaths every year. The use of synthetic pesticides against Culicidae leads to resistance and environmental concerns. Therefore, eco-friendly control tools are a priority. In this research, Nicandra physalodes-mediated synthesis of silver nanoparticles (Ag NPs) was conducted, in order to control larval populations of three important mosquito vectors, Anopheles stephensi, Aedes aegypti and Culex quinquefasciatus. Biofabricated Ag NPs were characterized using UV-vis spectrophotometry, XRD, FTIR spectroscopy, SEM, and TEM analyses. Ag NPs were highly toxic against the three mosquito vectors. Maximum efficacy was detected against A. stephensi (LC50=12.39μg/mL), followed by Ae. aegypti (LC50=13.61μg/mL) and Cx. quinquefasciatus (LC50=14.79μg/mL). Interestingly, Ag NPs were safer for the non-target aquatic organism Diplonychus indicus sharing the same aquatic habitats of mosquito larvae. LC50 and LC90 values were 1032.81 and 19,076.59μg/mL, respectively. Overall, our results highlight that N. physalodes-fabricated Ag NPs are a promising for development of eco-friendly larvicides against mosquito vectors, with negligible toxicity against non-target aquatic water bugs.

  9. An MRI-visible non-viral vector bearing GD2 single chain antibody for targeted gene delivery to human bone marrow mesenchymal stem cells.

    PubMed

    Pang, Pengfei; Wu, Chun; Shen, Min; Gong, Faming; Zhu, Kangshun; Jiang, Zaibo; Guan, Shouhai; Shan, Hong; Shuai, Xintao

    2013-01-01

    The neural ganglioside GD2 has recently been reported to be a novel surface marker that is only expressed on human bone marrow mesenchymal stem cells within normal marrow. In this study, an MRI-visible, targeted, non-viral vector for effective gene delivery to human bone marrow mesenchymal stem cells was first synthesized by attaching a targeting ligand, the GD2 single chain antibody (scAbGD2), to the distal ends of PEG-g-PEI-SPION. The targeted vector was then used to condense plasmid DNA to form nanoparticles showing stable small size, low cytotoxicity, and good biocompatibility. Based on a reporter gene assay, the transfection efficiency of targeting complex reached the highest value at 59.6% ± 4.5% in human bone marrow mesenchymal stem cells, which was higher than those obtained using nontargeting complex and lipofectamine/pDNA (17.7% ± 2.9% and 34.9% ± 3.6%, respectively) (P<0.01). Consequently, compared with the nontargeting group, more in vivo gene expression was observed in the fibrotic rat livers of the targeting group. Furthermore, the targeting capacity of scAbGD2-PEG-g-PEI-SPION was successfully verified in vitro by confocal laser scanning microscopy, Prussian blue staining, and magnetic resonance imaging. Our results indicate that scAbGD2-PEG-g-PEI-SPION is a promising MRI-visible non-viral vector for targeted gene delivery to human bone marrow mesenchymal stem cells.

  10. The synergistic effect of folate and RGD dual ligand of nanographene oxide on tumor targeting and photothermal therapy in vivo

    NASA Astrophysics Data System (ADS)

    Jang, Cheol; Lee, Jong Hyun; Sahu, Abhishek; Tae, Giyoong

    2015-11-01

    Effective delivery of nanoparticles to the target site is necessary for successful biomedical applications. Inefficient targeting is a major concern for nanomedicines in cancer therapy. Conjugation of multiple targeting ligands to the nanoparticle surface might further enhance the targeting efficiency by a co-operative effect of individual ligands. In this study, a dual ligand targeting nanographene oxide (nGO) was developed by non-covalent interaction with folate and cRGD functionalized pluronic, which allowed precise control of ligand number on the nGO surface and ensured stability under physiological conditions. The tumor targeting abilities of single and dual ligand decorated nGOs were evaluated in vitro by using KB cells, over-expressing folate and integrin αvβ3 receptors. In vitro cellular uptake analysis by flow cytometry and confocal laser scanning microscopy showed enhanced uptake of dual ligand modified nGO compared to any of the single ligand modified nGOs. The cellular uptake of dual targeted cRGD-FA-nGO was increased by 1.9 and 2.4 folds compared to single targeted cRGD-nGO or FA-nGO, respectively. The in vivo biodistribution experiment in a mouse xenograft model also confirmed the synergistic targeting effect of cRGD and folate dual functionalized nGO. A significantly higher tumor accumulation of cRGD-FA-nGO was observed compared to cRGD-nGO or FA-nGO. The higher tumor accumulation of dual targeted nGO resulted in complete ablation of tumor tissue through an enhanced photothermal effect by NIR laser irradiation. Therefore, co-functionalization of a nanoparticle by cRGD and folate is a potentially useful way to enhance the tumor targeting efficacy.Effective delivery of nanoparticles to the target site is necessary for successful biomedical applications. Inefficient targeting is a major concern for nanomedicines in cancer therapy. Conjugation of multiple targeting ligands to the nanoparticle surface might further enhance the targeting efficiency by a

  11. Dystrophin Delivery to Muscles of mdx Mice Using Lentiviral Vectors Leads to Myogenic Progenitor Targeting and Stable Gene Expression

    PubMed Central

    Kimura, En; Li, Sheng; Gregorevic, Paul; Fall, Brent M; Chamberlain, Jeffrey S

    2009-01-01

    To explore whether stable transduction of myogenic stem cells using lentiviral vectors could be of benefit for treating dystrophic muscles, we generated vectors expressing a functional microdystrophin/enhanced green fluorescence protein fusion (µDys/eGFP) gene. Lentiviral vector injection into neonatal mdx4cv muscles resulted in widespread and stable expression of dystrophin for at least 2 years. This expression resulted in a significant amelioration of muscle pathophysiology as assessed by a variety of histological and functional assays. To assess whether this long-term expression was accompanied by stable transduction of satellite cells, we harvested muscle mononuclear cells 1 year after vector injection. Up to 20% of the cultured myoblast colonies expressed the µDys/eGFP transgene following myotube formation. Furthermore, transplantation of the muscle mononuclear cells into secondary mdx4cv recipients showed their ability to regenerate dystrophin-expressing myofibers in vivo. The ability to isolate myogenic cells able to form dystrophin-positive myotubes or myofibers in vitro and in vivo >1 year postinjection indicates that the vectors stably transduced muscle satellite cells, or a progenitor of such cells, in neonatal mdx4cv muscles. These studies suggest that integrating lentiviral vectors have potential utility for gene therapy of muscular dystrophy. PMID:19888194

  12. Targeted chromosomal insertion of large DNA into the human genome by a fiber-modified high-capacity adenovirus-based vector system.

    PubMed

    Gonçalves, Manuel A F V; Holkers, Maarten; van Nierop, Gijsbert P; Wieringa, Roeland; Pau, Maria G; de Vries, Antoine A F

    2008-08-29

    A prominent goal in gene therapy research concerns the development of gene transfer vehicles that can integrate exogenous DNA at specific chromosomal loci to prevent insertional oncogenesis and provide for long-term transgene expression. Adenovirus (Ad) vectors arguably represent the most efficient delivery systems of episomal DNA into eukaryotic cell nuclei. The most advanced recombinant Ads lack all adenoviral genes. This renders these so-called high-capacity (hc) Ad vectors less cytotoxic/immunogenic than those only deleted in early regions and creates space for the insertion of large/multiple transgenes. The versatility of hcAd vectors is been increased by capsid modifications to alter their tropism and by the incorporation into their genomes of sequences promoting chromosomal insertion of exogenous DNA. Adeno-associated virus (AAV) can insert its genome into a specific human locus designated AAVS1. Trans- and cis-acting elements needed for this reaction are the AAV Rep78/68 proteins and Rep78/68-binding sequences, respectively. Here, we describe the generation, characterization and testing of fiber-modified dual hcAd/AAV hybrid vectors (dHVs) containing both these elements. Due to the inhibitory effects of Rep78/68 on Ad-dependent DNA replication, we deployed a recombinase-inducible gene switch to repress Rep68 synthesis during vector rescue and propagation. Flow cytometric analyses revealed that rep68-positive dHVs can be produced similarly well as rep68-negative control vectors. Western blot experiments and immunofluorescence microscopy analyses demonstrated transfer of recombinase-dependent rep68 genes into target cells. Studies in HeLa cells and in the dystrophin-deficient myoblasts from a Duchenne muscular dystrophy (DMD) patient showed that induction of Rep68 synthesis in cells transduced with fiber-modified and rep68-positive dHVs leads to increased stable transduction levels and AAVS1-targeted integration of vector DNA. These results warrant further

  13. Targeted therapy via oral administration of attenuated Salmonella expression plasmid-vectored Stat3-shRNA cures orthotopically transplanted mouse HCC

    PubMed Central

    Tian, Y; Guo, B; Jia, H; Ji, K; Sun, Y; Li, Y; Zhao, T; Gao, L; Meng, Y; Kalvakolanu, DV; Kopecko, DJ; Zhao, X; Zhang, L; Xu, D

    2013-01-01

    The development of RNA interference-based cancer gene therapies has been delayed due to the lack of effective tumor-targeting delivery systems. Attenuated Salmonella enterica serovar Typhimurium (S. Typhimurium) has a natural tropism for solid tumors. We report here the use of attenuated S. Typhimurium as a vector to deliver shRNA directly into tumor cells. Constitutively activated signal transducer and activator of transcription 3 (Stat3) is a key transcription factor involved in both hepatocellular carcinoma (HCC) growth and metastasis. In this study, attenuated S. Typhimurium was capable of delivering shRNA-expressing vectors to the targeted cancer cells and inducing RNA interference in vivo. More importantly, a single oral dose of attenuated S. Typhimurium carrying shRNA-expressing vectors targeting Stat3 induced remarkably delayed and reduced HCC (in 70% of mice). Cancer in these cured mice did not recur over 2 years following treatment. These data demonstrated that RNA interference combined with Salmonella as a delivery system may offer a novel clinical approach for cancer gene therapy. PMID:22555509

  14. Targeted therapy via oral administration of attenuated Salmonella expression plasmid-vectored Stat3-shRNA cures orthotopically transplanted mouse HCC.

    PubMed

    Tian, Y; Guo, B; Jia, H; Ji, K; Sun, Y; Li, Y; Zhao, T; Gao, L; Meng, Y; Kalvakolanu, D V; Kopecko, D J; Zhao, X; Zhang, L; Xu, D

    2012-06-01

    The development of RNA interference-based cancer gene therapies has been delayed due to the lack of effective tumor-targeting delivery systems. Attenuated Salmonella enterica serovar Typhimurium (S. Typhimurium) has a natural tropism for solid tumors. We report here the use of attenuated S. Typhimurium as a vector to deliver shRNA directly into tumor cells. Constitutively activated signal transducer and activator of transcription 3 (Stat3) is a key transcription factor involved in both hepatocellular carcinoma (HCC) growth and metastasis. In this study, attenuated S. Typhimurium was capable of delivering shRNA-expressing vectors to the targeted cancer cells and inducing RNA interference in vivo. More importantly, a single oral dose of attenuated S. Typhimurium carrying shRNA-expressing vectors targeting Stat3 induced remarkably delayed and reduced HCC (in 70% of mice). Cancer in these cured mice did not recur over 2 years following treatment. These data demonstrated that RNA interference combined with Salmonella as a delivery system may offer a novel clinical approach for cancer gene therapy.

  15. Analysis of Children's Perception of Triatomine Vectors of Chagas Disease through Drawings: Opportunities for Targeted Health Education

    PubMed Central

    Yevstigneyeva, Violetta; Camara-Mejia, Javier; Dumonteil, Eric

    2014-01-01

    Background Chagas disease is a tropical parasitic disease affecting about 10 million people, mostly in the Americas, and transmitted mainly by triatomine bugs. Insect vector control with indoor residual insecticides and the promotion of housing improvement is the main control intervention. The success of such interventions relies on their acceptance and appropriation by communities, which depends on their knowledge and perceptions of both the disease and the vector. In this study, we investigated school-aged children's knowledge and perception on triatomine vectors and Chagas disease to further understand how communities view this vector and the disease in Yucatan, Mexico. Methodology/Principal findings We performed an analysis of children's drawings on the theme of triatomines and their house in several rural villages, to explore in an open-ended manner their views, understanding and misconceptions. A total of 261 drawings were collected from children ages 6–12 from four villages. We found that children are very familiar with triatomine vectors, and know very well many aspects of their biology and ecology, and in particular their blood-feeding habits. On the other hand, their drawings suggest that the role of triatomines as vectors of a chronic and severe cardiac disease is less understood, and the main perceived health threat appears limited to the bite itself, as previously observed in adults. Conclusions/Significance These results have important implications for the specific design of future education materials and campaigns, and for the promotion of the inclusion of children in raising Chagas disease awareness in these endemic communities. PMID:25275321

  16. Lentiviral vector-mediated RNA interference targeted against prohibitin inhibits apoptosis of the retinoic acid-resistant acute promyelocytic leukemia cell line NB4-R1.

    PubMed

    Liu, Yanfeng; He, Pengcheng; Zhang, Mei; Wu, Di

    2012-12-01

    To investigate the possibility of prohibitin (PHB) inhibition by lentiviral vector-mediated RNA interference (RNAi) and its influence on cell apoptosis in the retinoic acid-resistant acute promyelocytic leukemia cell line NB4-R1, a lentiviral vector encoding a short hairpin RNA (shRNA) targeted against PHB (pGCSIL-GFP-PHB) was constructed and transfected into the packaging cells 293T, and the viral supernatant was collected to transfect NB4-R1 cells. Quantitative real-time fluorescent PCR and western blotting were used to detect the expression levels of PHB. Flow cytometry and detection of enzymatic activity of caspase-3 by western blotting were employed to examine cell apoptosis. Our results provide evidence that the lentiviral vector pGCSIL-GFP-PHB was constructed successfully, and the PHB mRNA and the protein expression inhibitory rates were 90.3 and 95.8%, respectively. When compared to the control group, the activity of caspase-3 decreased significantly, which showed a 57.3% downregulation, and the apoptosis rate was reduced by 44.6% (P<0.05). In conclusion, downregulation of the PHB gene may inhibit apoptosis of NB4-R1 cells, and it is speculated that this was at least partly due to the downregulation of caspase-3, and PHB may be a novel target for gene therapy for retinoic acid-resistant acute promyelocytic leukemia.

  17. [Regulatory mechanism of hormones of the pituitary-target gland axes in kidney-Yang deficiency based on a support vector machine model].

    PubMed

    Xiufeng, Wang; Lei, Zhang; Rongbo, Huang; Qinghua, Wu; Jianxin, Min; Na, Ma; Laicheng, Luo

    2015-04-01

    To study the development mechanism of kidney-Yang deficiency through the establishment of support vector machine models of relevant hormones of the pituitary-target gland axes in rats with kidney-Yang deficiency syndrome. The kidney-Yang deficiency rat model was created by intramuscular injection of hydrocortisone, and contents of the hormones of the pituitary-thyroid axis: thyroid stimulating hormone (TSH), 3,3',5-triiodothyronine (T3) and thyroxine (T4); hormones of the pituitary-adrenal gland axis: adrenocorticotropic hormone (ACTH) and cortisol (CORT); and hormones of the pituitary-gonadal axis: luteinizing hormone (LH), follicle-stimulating hormone (FSH), and testosterone (T), were determined in the early, middle, and advanced stages. Ten support vector regression (SVR) models of the hormones were established to analyze the mutual relationships among the hormones of the three axes. The feedback control action of the pituitary-adrenal axis began to lose efficacy from the middle stage of kidney-Yang deficiency. The contents all hormones of the three pituitary-target gland axes decreased in the advanced stage. Relative errors of the jackknife test of the SVR models all were less than 10%. Imbalances in mutual regulation among the hormones of the pituitary-target gland axes, especially loss of effectiveness of the pituitary-adrenal axis, is one pathogenesis of kidney-Yang deficiency. The SVR model can accurately reflect the complicated non-linear relationships among pituitary-target gland axes in rats with of kidney-Yang deficiency.

  18. Development of Nonviral Vectors Targeting the Brain as a Therapeutic Approach For Parkinson's Disease and Other Brain Disorders.

    PubMed

    Javed, Hayate; Menon, Sindhu A; Al-Mansoori, Karima M; Al-Wandi, Abdelmojib; Majbour, Nour K; Ardah, Mustafa T; Varghese, Shiji; Vaikath, Nishant N; Haque, M Emdadul; Azzouz, Mimoun; El-Agnaf, Omar Ma

    2016-04-01

    Parkinson's disease (PD) is a debilitating neurodegenerative disease characterized by tremor, rigidity, bradykinesia, and postural instability, for which there is no effective treatment available till date. Here, we report the development of nonviral vectors specific for neuronal cells that can deliver short interfering RNA (siRNA) against the α-synuclein gene (SNCA), and prevent PD-like symptoms both in vitro and in vivo. These vectors not only help siRNA duplexes cross the blood-brain barrier in mice, but also stabilize these siRNAs leading to a sustainable 60-90% knockdown of α-synuclein protein. Mice treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine rapidly develop PD-like symptoms which were significantly alleviated when SNCA was knocked down using our vectors. Together, our data not only confirm the central role of α-synuclein in the onset of PD, but also provide a proof of principle that these nonviral vectors can be used as novel tools to design effective strategies to combat central nervous system diseases.

  19. Application of Mutated miR-206 Target Sites Enables Skeletal Muscle-specific Silencing of Transgene Expression of Cardiotropic AAV9 Vectors

    PubMed Central

    Geisler, Anja; Schön, Christian; Größl, Tobias; Pinkert, Sandra; Stein, Elisabeth A; Kurreck, Jens; Vetter, Roland; Fechner, Henry

    2013-01-01

    Insertion of completely complementary microRNA (miR) target sites (miRTS) into a transgene has been shown to be a valuable approach to specifically repress transgene expression in non-targeted tissues. miR-122TS have been successfully used to silence transgene expression in the liver following systemic application of cardiotropic adeno-associated virus (AAV) 9 vectors. For miR-206–mediated skeletal muscle-specific silencing of miR-206TS–bearing AAV9 vectors, however, we found this approach failed due to the expression of another member (miR-1) of the same miR family in heart tissue, the intended target. We introduced single-nucleotide substitutions into the miR-206TS and searched for those which prevented miR-1–mediated cardiac repression. Several mutated miR-206TS (m206TS), in particular m206TS-3G, were resistant to miR-1, but remained fully sensitive to miR-206. All these variants had mismatches in the seed region of the miR/m206TS duplex in common. Furthermore, we found that some m206TS, containing mismatches within the seed region or within the 3′ portion of the miR-206, even enhanced the miR-206– mediated transgene repression. In vivo expression of m206TS-3G– and miR-122TS–containing transgene of systemically applied AAV9 vectors was strongly repressed in both skeletal muscle and the liver but remained high in the heart. Thus, site-directed mutagenesis of miRTS provides a new strategy to differentiate transgene de-targeting of related miRs. PMID:23439498

  20. Proposal of Path Following and Arrival Judgement Methods Using Target Vector for Teleoperation of a Mobile Robot on Uneven Ground by Image Pointing

    NASA Astrophysics Data System (ADS)

    Tamura, Sho; Maeyama, Shoichi

    Rescue robots have been actively developed since Hanshin-Awaji (Kobe) Earthquake. Recently, the rescue robot to reduce the risk of the secondary disaster on NBC terror and critical accident is also developed. For such a background, the development project of mobile RT system in the collapsed is started. This research also participates in this project. It is useful to use the image pointing for the control interface of the rescue robot because it can control the robot by the simple operation. However, the conventional method cannot work on a rough terrain. In this research, we propose the system which controls the robot to arrive the target position on the rough terrain. It is constructed the methods which put the destination into the vector, and control the 3D localizated robot to follow the vector. Finally, the proposed system is evaluated through experiments by remote control of a mobile robot in slope and cofirmed the feasibility.

  1. Adeno-associated viral vector serotypes 1 and 5 targeted to the neonatal rat and pig striatum induce widespread transgene expression in the forebrain.

    PubMed

    Kornum, Birgitte R; Stott, Simon R W; Mattsson, Bengt; Wisman, Liselijn; Ettrup, Anders; Hermening, Stephan; Knudsen, Gitte M; Kirik, Deniz

    2010-03-01

    Viral vector-mediated gene transfer has emerged as a powerful means to target transgene expression in the central nervous system. Here we characterized the efficacy of serotypes 1 and 5 recombinant adeno-associated virus (rAAV) vectors encoding green fluorescent protein (GFP) after stereotaxic delivery to the neonatal rat and minipig striatum. The efficiency of GFP expression and the phenotype of GFP-positive cells were assessed within the forebrain at different time points up to 12 months after surgery. Both rAAV1-GFP and rAAV5-GFP delivery resulted in transduction of the striatum as well as striatal input and output areas, including large parts of the cortex. In both species, rAAV5 resulted in a more widespread transgene expression compared to rAAV1. In neonatal rats, rAAV5 also transduced several other areas such as the olfactory bulbs, hippocampus, and septum. Phenotypic analysis of the GFP-positive cells, performed using immunohistochemistry and confocal microscopy, showed that most of the GFP-positive cells by either serotype were NeuN-positive neuronal profiles. The rAAV5 vector further displayed the ability to transduce non-neuronal cell types in both rats and pigs, albeit at a low frequency. Our results show that striatal delivery of rAAV5 vectors in the neonatal brain represents a useful tool to express genes of interest both in the basal ganglia and the neocortex. Furthermore, we apply, for the first time, viral vector-mediated gene transfer to the pig brain providing the opportunity to study effects of genetic manipulation in this non-primate large animal species. Finally, we generated an atlas of the Göttingen minipig brain for guiding future studies in this large animal species.

  2. Coupling of a bifunctional peptide R13 to OTMCS-PEI copolymer as a gene vector increases transfection efficiency and tumor targeting.

    PubMed

    Lv, Hui; Zhu, Qing; Liu, Kewu; Zhu, Manman; Zhao, Wenfang; Mao, Yuan; Liu, Kehai

    2014-01-01

    A degradable polyethylenimine (PEI) derivative coupled to a bifunctional peptide R13 was developed to solve the transfection efficiency versus cytotoxicity and tumor-targeting problems of PEI when used as a gene vector. We crossed-linked low molecular weight PEI with N-octyl-N-quaternary chitosan (OTMCS) to synthesize a degradable PEI derivative (OTMCS-PEI), and then used a bifunctional peptide, RGDC-TAT (49-57) called R13 to modify OTMCS-PEI so as to prepare a new gene vector, OTMCS-PEI-R13. This new gene vector was characterized by various physicochemical methods. Its cytotoxicity and gene transfection efficiency were also determined both in vitro and in vivo. The vector showed controlled degradation and excellent buffering capacity. The particle size of the OTMCS-PEI-R13/DNA complexes was around 150-250 nm and the zeta potential ranged from 10 mV to 30 mV. The polymer could protect plasmid DNA from being digested by DNase I at a concentration of 23.5 U DNase I/μg DNA. Further, the polymer was resistant to dissociation induced by 50% fetal bovine serum and 400 μg/mL sodium heparin. Compared with PEI 25 kDa, the OTMCS-PEI-R13/DNA complexes showed higher transfection efficiency both in vitro and in vivo. Further, compared with OTMCS-PEI, distribution of OTMCS-PEI-R13 at tumor sites was markedly enhanced, indicating the tumor-targeting specificity of R13. OTMCS-PEI-R13 could be a potential candidate as a safe and efficient gene delivery carrier for gene therapy.

  3. Coupling of a bifunctional peptide R13 to OTMCS-PEI copolymer as a gene vector increases transfection efficiency and tumor targeting

    PubMed Central

    Lv, Hui; Zhu, Qing; Liu, Kewu; Zhu, Manman; Zhao, Wenfang; Mao, Yuan; Liu, Kehai

    2014-01-01

    Background A degradable polyethylenimine (PEI) derivative coupled to a bifunctional peptide R13 was developed to solve the transfection efficiency versus cytotoxicity and tumor-targeting problems of PEI when used as a gene vector. Methods We crossed-linked low molecular weight PEI with N-octyl-N-quaternary chitosan (OTMCS) to synthesize a degradable PEI derivative (OTMCS-PEI), and then used a bifunctional peptide, RGDC-TAT (49–57) called R13 to modify OTMCS-PEI so as to prepare a new gene vector, OTMCS-PEI-R13. This new gene vector was characterized by various physicochemical methods. Its cytotoxicity and gene transfection efficiency were also determined both in vitro and in vivo. Results The vector showed controlled degradation and excellent buffering capacity. The particle size of the OTMCS-PEI-R13/DNA complexes was around 150–250 nm and the zeta potential ranged from 10 mV to 30 mV. The polymer could protect plasmid DNA from being digested by DNase I at a concentration of 23.5 U DNase I/μg DNA. Further, the polymer was resistant to dissociation induced by 50% fetal bovine serum and 400 μg/mL sodium heparin. Compared with PEI 25 kDa, the OTMCS-PEI-R13/DNA complexes showed higher transfection efficiency both in vitro and in vivo. Further, compared with OTMCS-PEI, distribution of OTMCS-PEI-R13 at tumor sites was markedly enhanced, indicating the tumor-targeting specificity of R13. Conclusion OTMCS-PEI-R13 could be a potential candidate as a safe and efficient gene delivery carrier for gene therapy. PMID:24648730

  4. First measurement of target and double spin asymmetries for e-vectorp-vector{yields}ep{pi}{sup 0} in the nucleon resonance region above the {delta}(1232)

    SciTech Connect

    Biselli, A. S.; Burkert, V. D.; Avakian, H.; Boiarinov, S.; Bosted, P.; Carman, D. S.; Degtyarenko, P. V.; Deur, A.; Egiyan, H.; Elouadrhiri, L.; Guo, L.; Gyurjyan, V.; Ito, M. M.; Kubarovsky, V.; Laget, J. M.; Mecking, B. A.; Mestayer, M. D.; Niczyporuk, B. B.; Nozar, M.; Sapunenko, V.

    2008-10-15

    The exclusive channel p-vectore-vector,e{sup '}p){pi}{sup 0} was studied in the first and second nucleon resonance regions in the Q{sup 2} range from 0.187 to 0.770 GeV{sup 2} at Jefferson Lab using the CEBAF Large Acceptance Spectrometer. Longitudinal target and beam-target asymmetries were extracted over a large range of center-of-mass angles of the {pi}{sup 0} and compared to the unitary isobar model MAID, the dynamic model by Sato and Lee, and the dynamic model DMT. A strong sensitivity to individual models was observed, in particular for the target asymmetry and in the higher invariant mass region. This data set, once included in the global fits of the above models, is expected to place strong constraints on the electrocoupling amplitudes A{sub 1/2} and S{sub 1/2} for the Roper resonance N(1400)P{sub 11} and the N(1535)S{sub 11} and N(1520)D{sub 13} states.

  5. Targeting expression of the leukemogenic PML-RARα fusion protein by lentiviral vector-mediated small interfering RNA results in leukemic cell differentiation and apoptosis.

    PubMed

    Ward, Simone V; Sternsdorf, Thomas; Woods, Niels-Bjarne

    2011-12-01

    Acute promyelocytic leukemia (APL) results from a chromosomal translocation that gives rise to the leukemogenic fusion protein PML-RARα (promyelocytic leukemia-retinoic acid α receptor). Differentiation of leukemic cells and complete remission of APL are achieved by treatment of patients with pharmacological doses of all-trans retinoic acid (ATRA), making APL a model disease for differentiation therapy. However, because patients are resistant to further treatment with ATRA on relapse, it is necessary to develop alternative treatment strategies to specifically target APL. We therefore sought to develop a treatment strategy based on lentiviral vector-mediated delivery of small interfering RNA (siRNA) that specifically targets the breakpoint region of PML-RARα. Unlike treatment with ATRA, which resulted in differentiation of leukemic NB4 cells, delivery of siRNA targeting PML-RARα into NB4 cells resulted in both differentiation and apoptosis, consistent with the specific knockdown of PML-RARα. Intraperitoneal injection of NB4 cells transduced with lentiviral vectors delivering PML-RARα-specific siRNA but not control siRNA prevented development of disease in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. Taken together, these results indicate that development of PML-RARα-specific siRNA may represent a promising treatment strategy for ATRA-resistant APL.

  6. Tumor-Specific Targeting With Modified Sindbis Viral Vectors: Evaluation with Optical Imaging and Positron Emission Tomography In Vivo

    PubMed Central

    Stelter, Lars; Tseng, Jen-Chieh; Torosjan, Armen; Levin, Brandi; Longo, Valerie A.; Pillarsetty, Nagavarakishore; Zanzonico, Pat; Meruelo, Daniel; Daniel, Steven M.

    2015-01-01

    Purpose Sindbis virus (SINV) infect tumor cells specifically and systemically throughout the body. Sindbis vectors are capable of expressing high levels of transduced suicide genes and thus efficiently produce enzymes for prodrug conversion in infected tumor cells. The ability to monitor suicide gene expression levels and viral load in patients, after administration of the vectors, would significantly enhance this tumor-specific therapeutic option. Procedures The tumor specificity of SINV is mediated by the 67-kDa laminin receptor (LR). We probed different cancer cell lines for their LR expression and, to determine the specific role of LR-expression in the infection cycle, used different molecular imaging strategies, such as bioluminescence, fluorescence molecular tomography, and positron emission tomography, to evaluate SINV-mediated infection in vitro and in vivo. Results All cancer cell lines showed a marked expression of LR. The infection rates of the SINV particles, however, differed significantly among the cell lines. Conclusion We used novel molecular imaging techniques to visualize vector delivery to different neoplatic cells. SINV infection rates proofed to be not solely dependent on cellular LR expression. Further studies need to evaluate the herein discussed ways of cellular infection and viral replication. PMID:22847302

  7. Cytotoxic-T-lymphocyte-mediated elimination of target cells transduced with engineered adeno-associated virus type 2 vector in vivo.

    PubMed

    Li, Chengwen; Hirsch, Matt; DiPrimio, Nina; Asokan, Aravind; Goudy, Kevin; Tisch, Roland; Samulski, R Jude

    2009-07-01

    A recent clinical trial in patients with hemophilia B has suggested that adeno-associated virus (AAV) capsid-specific cytotoxic T lymphocytes (CTLs) eliminated AAV-transduced hepatocytes and resulted in therapeutic failure. AAV capsids elicit a CTL response in animal models; however, these capsid-specific CTLs fail to kill AAV-transduced target cells in mice. To better model the human clinical trial data in mice, we introduced an immunodominant epitope derived from ovalbumin (OVA; SIINFEKL) into the AAV capsid and tested CTL-mediated killing of AAV2-transduced target tissues in vivo. Initially, in vitro experiments demonstrated both classical class I and cross-presentation of the OVA antigen, following endogenous expression or AAV2-OVA vector transduction, respectively. Furthermore, an OVA-specific CTL response was elicited after muscular or systemic injection of the AAV2-OVA vector. Finally, CTL reactivity was enhanced in mice with established SIINFEKL-specific immunity after AAV2-OVA/alpha1 anti-trypsin (AAT) administration. Most importantly, these OVA-specific CTLs decreased AAT expression in mice treated with AAV2-OVA/AAT vector that followed a time course mimicking uncoating kinetics of AAV2 transduction in OVA-immunized mice. These results demonstrate that AAV capsid-derived antigens elicit CD8(+) CTL reactivity, and these CTLs eliminated AAV-transduced target cells in mice. Notably, this model system can be exploited to study the kinetics of capsid presentation from different serotypes of AAV and permit the design of novel strategies to block CTL-mediated killing of AAV-transduced cells.

  8. Biophysical characterization of Acacia caesia-fabricated silver nanoparticles: effectiveness on mosquito vectors of public health relevance and impact on non-target aquatic biocontrol agents.

    PubMed

    Benelli, Giovanni; Kadaikunnan, Shine; Alharbi, Naiyf S; Govindarajan, Marimuthu

    2017-02-05

    Mosquito-borne diseases lead to serious public health concerns in tropical and sub-tropical countries worldwide, due to development of mosquito resistance to synthetic pesticides, non-target effects of pesticides, and socioeconomic reasons. Currently, green nanotechnology is a promising research field, showing a wide range of potential applications in vector control programs. The employ of natural products as reducing agents to fabricate insecticidal nanocomposites is gaining research attention worldwide, due to low costs and high effectiveness. Interestingly, biophysical features of green-synthesized nanoparticles strongly differ when different botanicals are employed for nanosynthesis. In this study, a cheap Acacia caesia leaf extract was employed to fabricate silver nanoparticles (Ag NPs) with ovicidal, larvicidal, and adulticidal toxicity against three mosquito vectors, Anopheles subpictus, Aedes albopictus, and Culex tritaeniorhynchus. Ag NPs were analyzed by various biophysical methods, including spectroscopy (UV-visible spectrophotometry, XRD, FTIR, EDX) and microscopy (SEM, TEM, AFM) techniques. High acute larvicidal potential was observed against larvae of An. subpictus (LC50 = 10.33 μg/ml), Ae. albopictus (LC50 = 11.32 μg/ml), and Cx. tritaeniorhynchus (LC50 = 12.35 μg/ml). Ag NPs completely inhibited egg hatchability on three vectors at 60, 75, and 90 μg/ml, respectively. In adulticidal assays, LD50 values were 18.66, 20.94, and 22.63 μg/ml. If compared to mosquito larvae, Ag NPs were safer to three non-target aquatic biocontrol agents, with LC50 ranging from 684 to 2245 μg/ml. Overall, our study highlights the potential of A. caesia as an abundant and cheap bioresource to fabricate biogenic Ag NPs effective against mosquito young instars and adults, with moderate impact on non-target aquatic biocontrol agents.

  9. A new model of multi-visceral and bone metastatic prostate cancer with perivascular niche targeting by a novel endothelial specific adenoviral vector.

    PubMed

    Lu, Zhi Hong; Kaliberov, Sergey; Sohn, Rebecca E; Kaliberova, Lyudmila; Du, Yingqiu; Prior, Julie L; Leib, Daniel J; Chauchereau, Anne; Sehn, Jennifer K; Curiel, David T; Arbeit, Jeffrey M

    2017-01-17

    While modern therapies for metastatic prostate cancer (PCa) have improved survival they are associated with an increasingly prevalent entity, aggressive variant PCa (AVPCa), lacking androgen receptor (AR) expression, enriched for cancer stem cells (CSCs), and evidencing epithelial-mesenchymal plasticity with a varying extent of neuroendocrine transdifferentiation. Parallel work revealed that endothelial cells (ECs) create a perivascular CSC niche mediated by juxtacrine and membrane tethered signaling. There is increasing interest in pharmacological metastatic niche targeting, however, targeted access has been impossible. Here, we discovered that the Gleason 7 derived, androgen receptor negative, IGR-CaP1 cell line possessed some but not all of the molecular features of AVPCa. Intracardiac injection into NOD/SCID/IL2Rg -/- (NSG) mice produced a completely penetrant bone, liver, adrenal, and brain metastatic phenotype; noninvasively and histologically detectable at 2 weeks, and necessitating sacrifice 4-5 weeks post injection. Bone metastases were osteoblastic, and osteolytic. IGR-CaP1 cells expressed the neuroendocrine marker synaptophysin, near equivalent levels of vimentin and e-cadherin, all of the EMT transcription factors, and activation of NOTCH and WNT pathways. In parallel, we created a new triple-targeted adenoviral vector containing a fiber knob RGD peptide, a hexon mutation, and an EC specific ROBO4 promoter (Ad.RGD.H5/3.ROBO4). This vector was expressed in metastatic microvessels tightly juxtaposed to IGR-CaP1 cells in bone and visceral niches. Thus, the combination of IGR-CaP1 cells and NSG mice produces a completely penetrant metastatic PCa model emulating end-stage human disease. In addition, the metastatic niche access provided by our novel Ad vector could be therapeutically leveraged for future disease control or cure.

  10. Cytotoxic-T-Lymphocyte-Mediated Elimination of Target Cells Transduced with Engineered Adeno-Associated Virus Type 2 Vector In Vivo▿

    PubMed Central

    Li, Chengwen; Hirsch, Matt; DiPrimio, Nina; Asokan, Aravind; Goudy, Kevin; Tisch, Roland; Samulski, R. Jude

    2009-01-01

    A recent clinical trial in patients with hemophilia B has suggested that adeno-associated virus (AAV) capsid-specific cytotoxic T lymphocytes (CTLs) eliminated AAV-transduced hepatocytes and resulted in therapeutic failure. AAV capsids elicit a CTL response in animal models; however, these capsid-specific CTLs fail to kill AAV-transduced target cells in mice. To better model the human clinical trial data in mice, we introduced an immunodominant epitope derived from ovalbumin (OVA; SIINFEKL) into the AAV capsid and tested CTL-mediated killing of AAV2-transduced target tissues in vivo. Initially, in vitro experiments demonstrated both classical class I and cross-presentation of the OVA antigen, following endogenous expression or AAV2-OVA vector transduction, respectively. Furthermore, an OVA-specific CTL response was elicited after muscular or systemic injection of the AAV2-OVA vector. Finally, CTL reactivity was enhanced in mice with established SIINFEKL-specific immunity after AAV2-OVA/α1 anti-trypsin (AAT) administration. Most importantly, these OVA-specific CTLs decreased AAT expression in mice treated with AAV2-OVA/AAT vector that followed a time course mimicking uncoating kinetics of AAV2 transduction in OVA-immunized mice. These results demonstrate that AAV capsid-derived antigens elicit CD8+ CTL reactivity, and these CTLs eliminated AAV-transduced target cells in mice. Notably, this model system can be exploited to study the kinetics of capsid presentation from different serotypes of AAV and permit the design of novel strategies to block CTL-mediated killing of AAV-transduced cells. PMID:19369348

  11. Long-lasting insecticide-treated house screens and targeted treatment of productive breeding-sites for dengue vector control in Acapulco, Mexico

    PubMed Central

    Che-Mendoza, Azael; Guillermo-May, Guillermo; Herrera-Bojórquez, Josué; Barrera-Pérez, Mario; Dzul-Manzanilla, Felipe; Gutierrez-Castro, Cipriano; Arredondo-Jiménez, Juan I.; Sánchez-Tejeda, Gustavo; Vazquez-Prokopec, Gonzalo; Ranson, Hilary; Lenhart, Audrey; Sommerfeld, Johannes; McCall, Philip J.; Kroeger, Axel; Manrique-Saide, Pablo

    2015-01-01

    Background Long-lasting insecticidal net screens (LLIS) fitted to domestic windows and doors in combination with targeted treatment (TT) of the most productive Aedes aegypti breeding sites were evaluated for their impact on dengue vector indices in a cluster-randomised trial in Mexico between 2011 and 2013. Methods Sequentially over 2 years, LLIS and TT were deployed in 10 treatment clusters (100 houses/cluster) and followed up over 24 months. Cross-sectional surveys quantified infestations of adult mosquitoes, immature stages at baseline (pre-intervention) and in four post-intervention samples at 6-monthly intervals. Identical surveys were carried out in 10 control clusters that received no treatment. Results LLIS clusters had significantly lower infestations compared to control clusters at 5 and 12 months after installation, as measured by adult (male and female) and pupal-based vector indices. After addition of TT to the intervention houses in intervention clusters, indices remained significantly lower in the treated clusters until 18 (immature and adult stage indices) and 24 months (adult indices only) post-intervention. Conclusions These safe, simple affordable vector control tools were well-accepted by study participants and are potentially suitable in many regions at risk from dengue worldwide. PMID:25604761

  12. Long-lasting insecticide-treated house screens and targeted treatment of productive breeding-sites for dengue vector control in Acapulco, Mexico.

    PubMed

    Che-Mendoza, Azael; Guillermo-May, Guillermo; Herrera-Bojórquez, Josué; Barrera-Pérez, Mario; Dzul-Manzanilla, Felipe; Gutierrez-Castro, Cipriano; Arredondo-Jiménez, Juan I; Sánchez-Tejeda, Gustavo; Vazquez-Prokopec, Gonzalo; Ranson, Hilary; Lenhart, Audrey; Sommerfeld, Johannes; McCall, Philip J; Kroeger, Axel; Manrique-Saide, Pablo

    2015-02-01

    Long-lasting insecticidal net screens (LLIS) fitted to domestic windows and doors in combination with targeted treatment (TT) of the most productive Aedes aegypti breeding sites were evaluated for their impact on dengue vector indices in a cluster-randomised trial in Mexico between 2011 and 2013. Sequentially over 2 years, LLIS and TT were deployed in 10 treatment clusters (100 houses/cluster) and followed up over 24 months. Cross-sectional surveys quantified infestations of adult mosquitoes, immature stages at baseline (pre-intervention) and in four post-intervention samples at 6-monthly intervals. Identical surveys were carried out in 10 control clusters that received no treatment. LLIS clusters had significantly lower infestations compared to control clusters at 5 and 12 months after installation, as measured by adult (male and female) and pupal-based vector indices. After addition of TT to the intervention houses in intervention clusters, indices remained significantly lower in the treated clusters until 18 (immature and adult stage indices) and 24 months (adult indices only) post-intervention. These safe, simple affordable vector control tools were well-accepted by study participants and are potentially suitable in many regions at risk from dengue worldwide. © The author 2015. The World Health Organization has granted Oxford University Press permission for the reproduction of this article.

  13. Systemic correction of the muscle disorder glycogen storage disease type II after hepatic targeting of a modified adenovirus vector encoding human acid-α-glucosidase

    PubMed Central

    Amalfitano, A.; McVie-Wylie, A. J.; Hu, H.; Dawson, T. L.; Raben, N.; Plotz, P.; Chen, Y. T.

    1999-01-01

    This report demonstrates that a single intravenous administration of a gene therapy vector can potentially result in the correction of all affected muscles in a mouse model of a human genetic muscle disease. These results were achieved by capitalizing both on the positive attributes of modified adenovirus-based vectoring systems and receptor-mediated lysosomal targeting of enzymes. The muscle disease treated, glycogen storage disease type II, is a lysosomal storage disorder that manifests as a progressive myopathy, secondary to massive glycogen accumulations in the skeletal and/or cardiac muscles of affected individuals. We demonstrated that a single intravenous administration of a modified Ad vector encoding human acid α-glucosidase (GAA) resulted in efficient hepatic transduction and secretion of high levels of the precursor GAA proenzyme into the plasma of treated animals. Subsequently, systemic distribution and uptake of the proenzyme into the skeletal and cardiac muscles of the GAA-knockout mouse was confirmed. As a result, systemic decreases (and correction) of the glycogen accumulations in a variety of muscle tissues was demonstrated. This model can potentially be expanded to include the treatment of other lysosomal enzyme disorders. Lessons learned from systemic genetic therapy of muscle disorders also should have implications for other muscle diseases, such as the muscular dystrophies. PMID:10430861

  14. Plant microRNA-Target Interaction Identification Model Based on the Integration of Prediction Tools and Support Vector Machine

    PubMed Central

    Meng, Jun; Shi, Lin; Luan, Yushi

    2014-01-01

    Background Confident identification of microRNA-target interactions is significant for studying the function of microRNA (miRNA). Although some computational miRNA target prediction methods have been proposed for plants, results of various methods tend to be inconsistent and usually lead to more false positive. To address these issues, we developed an integrated model for identifying plant miRNA–target interactions. Results Three online miRNA target prediction toolkits and machine learning algorithms were integrated to identify and analyze Arabidopsis thaliana miRNA-target interactions. Principle component analysis (PCA) feature extraction and self-training technology were introduced to improve the performance. Results showed that the proposed model outperformed the previously existing methods. The results were validated by using degradome sequencing supported Arabidopsis thaliana miRNA-target interactions. The proposed model constructed on Arabidopsis thaliana was run over Oryza sativa and Vitis vinifera to demonstrate that our model is effective for other plant species. Conclusions The integrated model of online predictors and local PCA-SVM classifier gained credible and high quality miRNA-target interactions. The supervised learning algorithm of PCA-SVM classifier was employed in plant miRNA target identification for the first time. Its performance can be substantially improved if more experimentally proved training samples are provided. PMID:25051153

  15. Priorities and needs for research on urban interventions targeting vector-borne diseases: rapid review of scoping and systematic reviews.

    PubMed

    Bermudez-Tamayo, Clara; Mukamana, Olive; Carabali, Mabel; Osorio, Lyda; Fournet, Florence; Dabiré, Kounbobr Roch; Turchi Marteli, Celina; Contreras, Adolfo; Ridde, Valéry

    2016-12-01

    This paper highlights the critical importance of evidence on vector-borne diseases (VBD) prevention and control interventions in urban settings when assessing current and future needs, with a view to setting policy priorities that promote inclusive and equitable urban health services. Research should produce knowledge about policies and interventions that are intended to control and prevent VBDs at the population level and to reduce inequities. Such interventions include policy, program, and resource distribution approaches that address the social determinants of health and exert influence at organizational and system levels.

  16. Correlations between household occupancy and malaria vector biting risk in rural Tanzanian villages: implications for high-resolution spatial targeting of control interventions.

    PubMed

    Kaindoa, Emmanuel W; Mkandawile, Gustav; Ligamba, Godfrey; Kelly-Hope, Louise A; Okumu, Fredros O

    2016-04-12

    Fine-scale targeting of interventions is increasingly important where epidemiological disease profiles depict high geographical stratifications. This study verified correlations between household biomass and mosquito house-entry using experimental hut studies, and then demonstrated how geographical foci of mosquito biting risk can be readily identified based on spatial distributions of household occupancies in villages. A controlled 4 × 4 Latin square experiment was conducted in rural Tanzania, in which no, one, three or six adult male volunteers slept under intact bed nets, in experimental huts. Mosquitoes entering the huts were caught using exit interception traps on eaves and windows. Separately, monthly mosquito collections were conducted in 96 randomly selected households in three villages using CDC light traps between March-2012 and November-2013. The number of people sleeping in the houses and other household and environmental characteristics were recorded. ArcGIS 10 (ESRI-USA) spatial analyst tool, Gi* Ord Statistic was used to analyse clustering of vector densities and household occupancy. The densities of all mosquito genera increased in huts with one, three or six volunteers, relative to huts with no volunteers, and direct linear correlations within tested ranges (P < 0.001). Significant geographical clustering of indoor densities of malaria vectors, Anopheles arabiensis and Anopheles funestus, but not Culex or Mansonia species occurred in locations where households with highest occupancy were also most clustered (Gi* P ≤ 0.05, and Gi* Z-score ≥ 1.96). This study demonstrates strong correlations between household occupancy and malaria vector densities in households, but also spatial correlations of these variables within and between villages in rural southeastern Tanzania. Fine-scale clustering of indoor densities of vectors within and between villages occurs in locations where houses with highest occupancy are also clustered. The study indicates

  17. Adenoviral vectors modified by heparin-polyethyleneimine nanogels enhance targeting to the lung and show therapeutic potential for pulmonary metastasis in vivo.

    PubMed

    Wei, Wei; Mu, Yandong; Li, XiaoPeng; Gou, MaLing; Zhang, HaiLong; Luo, ShunTao; Men, Ke; Mao, YongQiu; Qian, ZhiYong; Yang, Li

    2011-12-01

    Polyethyleneimine (PEI) is a well-known cationic polymer that has previously been shown to have significant potential to deliver genes in vitro and in vivo. However, PEI is non-degradable and exhibits a high cytotoxicity as its molecular weight increases. The clinical application for systemic administration of adenoviral (Ad) vectors is limited, as these vectors do not efficiently penetrate solid tumor masses due to a common deficiency of Coxsackie Adenovirus Receptor (CAR) on the tumor surface. In this study, we conjugated low molecular weight PEI (Mn = 1,800) to heparin (Mn = 4,000-6,000) to create a new type of cationic degradable nanogel (HPEI) that was then used to modify Ad vectors. The resulting HPEI-Ad complexes were used to infect CT26 and HeLa cells in vitro. Additionally, the HPEI-Ad complexes were administrated in vivo via intravenous injection, and tissue distribution was assessed using luciferase assays; the therapeutic potential of HPEI-Ad complexes for pulmonary metastasis mediated by CT26 cells was also investigated. In vitro, HPEI-Ad complexes enhanced the transfection efficiency in CT26 cells, reaching 36.3% compared with 0.1% of the native adenovirus. In vivo, HPEI-Ad complexes exhibited greater affinity for lung tissue than the native adenovirus and effectively inhibited the growth of pulmonary metastases mediated by CT26 cells. Our results indicate that Ad vectors modified by HPEI nanogels to form HPEI-Ad complexes enhanced transfection efficiency in CT26 cells that lacked CAR, targeted to the lung and demostrated a potential therapy for pulmonary metastasis.

  18. Larvivorous fish in wells target the malaria vector sibling species of the Anopheles culicifacies complex in villages in Karnataka, India.

    PubMed

    Ghosh, S K; Tiwari, S N; Sathyanarayan, T S; Sampath, T R R; Sharma, V P; Nanda, Nutan; Joshi, Hema; Adak, T; Subbarao, S K

    2005-02-01

    Malaria was a major problem in a sericulture area of Karnataka, south India, where Anopheles culicifacies s.l. and A. fluviatilis s.l. were considered to be the main vectors. Sibling species complexes of these two species were analysed in three ecologically different villages. Among A. culicifacies, only sibling species A and B were found. In Puram, a village with 22 wells, species A predominated; species B predominated in a village with four wells and a stream, and in a village with a stream and no wells. Poecilia reticulata fish were introduced into all wells and streams in the villages, and after one year no vectors were found in Puram, and all, or nearly all, A. culicifacies were species B in the other two villages. All A. fluviatilis belonged to the sibling species T. Blood meal analysis indicated that a few of the A. culicifacies collected had fed on humans while all the A. fluviatilis had fed on bovines. Before the introduction of fish, the annual parasite incidence for malaria was high in Puram, but much lower in the other two villages. From 1998 (over one year after release of fish) until 2003, no malaria cases were detected in the three villages.

  19. A tetracycline-regulated cell line produces high-titer lentiviral vectors that specifically target dendritic cells.

    PubMed

    Bryson, Paul D; Zhang, Chupei; Lee, Chi-Lin; Wang, Pin

    2013-06-19

    Lentiviral vectors (LVs) are a powerful means of delivering genetic material to many types of cells. Because of safety concerns associated with these HIV-1 derived vectors, producing large quantities of LVs is challenging. In this paper, we report a method for producing high titers of self-inactivating LVs. We retrovirally transduce the tet-off stable producer cell line GPR to generate a cell line, GPRS, which can express all the viral components, including a dendritic cell-specific glycoprotein, SVGmu. Then, we use concatemeric DNA transfection to transfect the LV transfer plasmid encoding a reporter gene GFP in combination with a selectable marker. Several of the resulting clones can produce LV at a titer 10-fold greater than what we achieve with transient transfection. Plus, these viruses efficiently transduce dendritic cells in vitro and generate a strong T cell immune response to our reporter antigen. This method may be a good option for producing strong LV-based vaccines for clinical studies of cancer or infectious diseases.

  20. Development of a Non-integrating Rev-dependent Lentiviral Vector Carrying Diphtheria Toxin A Chain and Human TRAF6 to Target HIV Reservoirs

    PubMed Central

    Wang, Zhirui; Tang, Zhongwei; Zheng, Yanfang; Yu, Dongyang; Spear, Mark; Iyer, Subashini R.; Bishop, Barney; Wu, Yuntao

    2010-01-01

    Persistence of HIV despite highly active antiretroviral therapy (HAART) is a lasting challenge to virus eradication. To develop a strategy complementary to HAART, we constructed a series of Rev-dependent lentiviral vectors carrying diphtheria toxin A chain (DT-A) and its attenuated mutants, as well as human TRAF6. Expression of these suicide genes following delivery through viral particles is dependent on Rev, which exists only in infected cells. Among these toxins, DT-A has been known to trigger cell death with as little as a single molecule, whereas two of the attenuated mutants in this study, DT-A(176) and DT-A(ΔN), were well-tolerated by cells at low levels. TRAF6 induced apoptosis only with persistent overexpression. Thus, these suicide genes, which induce cell death at different expression levels, offer a balance between efficacy and safety. To minimize possible mutagenesis introduced by retroviral integration in non-target cells, we further developed a non-integrating Rev-dependent (NIRD) lentiviral vector to deliver these genes. In addition, we constructed a DT-A-resistant human cell line by introducing a human elongation factor 2 (EF-2) mutant into HEK293T cells. This allowed us to manufacture the first high-titer NIRD lentiviral particles carrying DT-A to target HIV-positive cells. PMID:20410930

  1. One-pot biogenic fabrication of silver nanocrystals using Quisqualis indica: Effectiveness on malaria and Zika virus mosquito vectors, and impact on non-target aquatic organisms.

    PubMed

    Govindarajan, Marimuthu; Vijayan, Periasamy; Kadaikunnan, Shine; Alharbi, Naiyf S; Benelli, Giovanni

    2016-09-01

    Currently, mosquito vector control is facing a number of key challenges, including the rapid development of resistance to synthetic pesticides and the recent spread of aggressive arbovirus outbreaks. The biosynthesis of silver nanoparticles (AgNPs) is currently considered an environmental friendly alternative to the employ of pyrethroids, carbamates and microbial agents (e.g. Bacillus thuringiensis var. israelensis), since AgNPs are easy to produce, effective and stable in the aquatic environment. However, their biophysical features showed wide variations according to the botanical agent using for the green synthesis, outlining the importance of screening local floral resources used as reducing and stabilizing agents. In this study, we focused on the biophysical properties and the mosquitocidal action of Quisqualis indica-fabricated AgNPs. AgNPs were characterized using spectroscopic (UV, FTIR, XRD) and microscopic (AFM, SEM, TEM and EDX) techniques. AFM, SEM and TEM confirmed the synthesis of poly-dispersed AgNPs with spherical shape and size ranging from 1 to 30nm. XRD shed light on the crystalline structure of these AgNPs. The acute toxicity of Quisqualis indica extract and AgNPs was evaluated against malaria, arbovirus, and filariasis vectors, Anopheles stephensi, Aedes aegypti and Culex quinquefasciatus, as well as on three important non-target aquatic organisms. The Q. indica leaf extract showed moderate larvicidal effectiveness on Cx. quinquefasciatus (LC50=220.42), Ae. aegypti (LC50=203.63) and An. stephensi (LC50=185.98). Q. indica-fabricated AgNPs showed high toxicity against Cx. quinquefasciatus (LC50=14.63), Ae. aegypti (LC50=13.55) and An. stephensi (LC50=12.52), respectively. Notably, Q. indica-synthesized AgNPs were moderately toxic to non-target aquatic mosquito predators Anisops bouvieri (LC50=653.05μg/mL), Diplonychus indicus (LC50=860.94μg/mL) and Gambusia affinis (LC50=2183.16μg/mL), if compared to the targeted mosquitoes. Overall, the

  2. pH-responsive three-layered PEGylated polyplex micelle based on a lactosylated ABC triblock copolymer as a targetable and endosome-disruptive nonviral gene vector.

    PubMed

    Oishi, Motoi; Kataoka, Kazunori; Nagasaki, Yukio

    2006-01-01

    Nonviral vectors for gene therapy have recently received an increased impetus because of the inherent safety problems of the viral vectors, while their transfection efficiency is generally low compared to the viral vectors. The lack of the ability to escape from the endosomal compartments is believed to be one of the critical barriers to the intracellular delivery of noviral gene vectors. This study was devoted to the design and preparation of a novel ABC triblock copolymer for constructing a pH-responsive and targetable nonviral gene vector. The copolymer, lactosylated poly(ethylene glycol)-block-poly(silamine)-block-poly[2-(N,N-dimethylamino)ethyl methacrylate] (Lac-PEG-PSAO-PAMA), consists of lactosylated poly(ethylene glycol) (A-segment), a pH-responsive polyamine segment (B-segment), and a DNA-condensing polyamine segment (C-segment). The Lac-PEG-PSAO-PAMA spontaneously associated with plasmid DNA (pDNA) to form three-layered polyplex micelles with a PAMA/pDNA polyion complex (PIC) core, an uncomplexed PSAO inner shell, and a lactosylated PEG outer shell, as confirmed by 1H NMR spectroscopy. Under physiological conditions, the Lac-PEG-PSAO-PAMA/pDNA polyplex micelles prepared at an N/P (number of amino groups in the copolymer/number of phosphate groups in pDNA) ratio above 3 were found to be able to condense pDNA, thus adopting a relatively small size (< 150 nm) and an almost neutral surface charge (zeta approximately +5 mV). The micelle underwent a pH-induced size variation (pH = 7.4, 132.6 nm --> pH = 4.0, 181.8 nm) presumably due to the conformational changes (globule-rod transition) of the uncomplexed PSAO chain in response to pH, leading to swelling of the free PSAO inner shell at lowered pH while retaining the condensed pDNA in the PAMA/pDNA PIC core. Furthermore, the micelles exhibited a specific cellular uptake into HuH-7 cells (hepatocytes) through asialoglycoprotein (ASGP) receptor-mediated endocytosis and achieved a far more efficient transfection

  3. Antibody-mediated targeted gene transfer to NMDA NR1-containing neurons in rat neocortex by helper virus-free HSV-1 vector particles containing a chimeric HSV-1 glycoprotein C-staphylococcus A protein.

    PubMed

    Cao, Haiyan; Zhang, Guo-Rong; Geller, Alfred I

    2010-09-10

    Because of the heterogeneous cellular composition of the brain, and especially the forebrain, cell type-specific expression will benefit many potential applications of direct gene transfer. The two prevalent approaches for achieving cell type-specific expression are using a cell type-specific promoter or targeting gene transfer to a specific cell type. Targeted gene transfer with Herpes Simplex Virus (HSV-1) vectors modifies glycoprotein C (gC) to replace the heparin binding domain, which binds to many cell types, with a binding activity for a specific cell surface protein. We previously reported targeted gene transfer to nigrostriatal neurons using chimeric gC-glial cell line-derived neurotrophic factor or gC-brain-derived neurotrophic factor protein. Unfortunately, this approach is limited to cells that express the cognate receptor for either neurotrophic factor. Thus, a general strategy for targeting gene transfer to many different types of neurons is desirable. Antibody-mediated targeted gene transfer has been developed for targeting specific virus vectors to specific peripheral cell types; a specific vector particle protein is modified to contain the Staphylococcus A protein ZZ domain, which binds immunoglobulin (Ig) G. Here, we report antibody-mediated targeted gene transfer of HSV-1 vectors to a specific type of forebrain neuron. We constructed a chimeric gC-ZZ protein, and showed this protein is incorporated into vector particles and binds Ig G. Complexes of these vector particles and an antibody to the NMDA receptor NR1 subunit supported targeted gene transfer to NR1-containing neocortical neurons in the rat brain, with long-term (2 months) expression.

  4. Retroviral Replicating Vectors Deliver Cytosine Deaminase Leading to Targeted 5-Fluorouracil-Mediated Cytotoxicity in Multiple Human Cancer Types.

    PubMed

    Twitty, Chris G; Diago, Oscar R; Hogan, Daniel J; Burrascano, Cindy; Ibanez, Carlos E; Jolly, Douglas J; Ostertag, Derek

    2016-02-01

    Toca 511 is a modified retroviral replicating vector based on Moloney γ-retrovirus with an amphotropic envelope. As an investigational cancer treatment, Toca 511 preferentially infects cancer cells without direct cell lysis and encodes an enhanced yeast cytosine deaminase that converts the antifungal drug 5-fluorocytosine to the anticancer drug, 5-fluorouracil. A panel of established human cancer cell lines, derived from glioblastoma, colon, and breast cancer tissue, was used to evaluate parameters critical for effective anticancer activity. Gene transfer, cytosine deaminase production, conversion of 5-fluorocytosine to 5-fluorouracil, and subsequent cell killing occurred in all lines tested. We observed >50% infection within 25 days in all lines and 5-fluorocytosine LD50 values between 0.02 and 6 μg/ml. Although we did not identify a small number of key criteria, these studies do provide a straightforward approach to rapidly gauge the probability of a Toca 511 and 5-fluorocytosine treatment effect in various cancer indications: a single MTS assay of maximally infected cancer cell lines to determine 5-fluorocytosine LD50. The data suggest that, although there can be variation in susceptibility to Toca 511 and 5-fluorocytosine because of multiple mechanistic factors, this therapy may be applicable to a broad range of cancer types and individuals.

  5. Retroviral Replicating Vectors Deliver Cytosine Deaminase Leading to Targeted 5-Fluorouracil-Mediated Cytotoxicity in Multiple Human Cancer Types

    PubMed Central

    Twitty, Chris G.; Diago, Oscar R.; Hogan, Daniel J.; Burrascano, Cindy; Ibanez, Carlos E.; Jolly, Douglas J.; Ostertag, Derek

    2016-01-01

    Toca 511 is a modified retroviral replicating vector based on Moloney γ-retrovirus with an amphotropic envelope. As an investigational cancer treatment, Toca 511 preferentially infects cancer cells without direct cell lysis and encodes an enhanced yeast cytosine deaminase that converts the antifungal drug 5-fluorocytosine to the anticancer drug, 5-fluorouracil. A panel of established human cancer cell lines, derived from glioblastoma, colon, and breast cancer tissue, was used to evaluate parameters critical for effective anticancer activity. Gene transfer, cytosine deaminase production, conversion of 5-fluorocytosine to 5-fluorouracil, and subsequent cell killing occurred in all lines tested. We observed >50% infection within 25 days in all lines and 5-fluorocytosine LD50 values between 0.02 and 6 μg/ml. Although we did not identify a small number of key criteria, these studies do provide a straightforward approach to rapidly gauge the probability of a Toca 511 and 5-fluorocytosine treatment effect in various cancer indications: a single MTS assay of maximally infected cancer cell lines to determine 5-fluorocytosine LD50. The data suggest that, although there can be variation in susceptibility to Toca 511 and 5-fluorocytosine because of multiple mechanistic factors, this therapy may be applicable to a broad range of cancer types and individuals. PMID:26467507

  6. Singular vector-based targeted observations of chemical constituents: description and first application of the EURAD-IM-SVA v1.0

    NASA Astrophysics Data System (ADS)

    Goris, N.; Elbern, H.

    2015-12-01

    Measurements of the large-dimensional chemical state of the atmosphere provide only sparse snapshots of the state of the system due to their typically insufficient temporal and spatial density. In order to optimize the measurement configurations despite those limitations, the present work describes the identification of sensitive states of the chemical system as optimal target areas for adaptive observations. For this purpose, the technique of singular vector analysis (SVA), which has proven effective for targeted observations in numerical weather prediction, is implemented in the EURAD-IM (EURopean Air pollution and Dispersion - Inverse Model) chemical transport model, yielding the EURAD-IM-SVA v1.0. Besides initial values, emissions are investigated as critical simulation controlling targeting variables. For both variants, singular vectors are applied to determine the optimal placement for observations and moreover to quantify which chemical compounds have to be observed with preference. Based on measurements of the airship based ZEPTER-2 campaign, the EURAD-IM-SVA v1.0 has been evaluated by conducting a comprehensive set of model runs involving different initial states and simulation lengths. For the sake of brevity, we concentrate our attention on the following chemical compounds, O3, NO, NO2, HCHO, CO, HONO, and OH, and focus on their influence on selected O3 profiles. Our analysis shows that the optimal placement for observations of chemical species is not entirely determined by mere transport and mixing processes. Rather, a combination of initial chemical concentrations, chemical conversions, and meteorological processes determines the influence of chemical compounds and regions. We furthermore demonstrate that the optimal placement of observations of emission strengths is highly dependent on the location of emission sources and that the benefit of including emissions as target variables outperforms the value of initial value optimization with growing

  7. The enzyme 3-hydroxykynurenine transaminase as potential target for 1,2,4-oxadiazoles with larvicide activity against the dengue vector Aedes aegypti.

    PubMed

    Oliveira, Vanessa S; Pimenteira, Cecília; da Silva-Alves, Diana C B; Leal, Laylla L L; Neves-Filho, Ricardo A W; Navarro, Daniela M A F; Santos, Geanne K N; Dutra, Kamilla A; dos Anjos, Janaína V; Soares, Thereza A

    2013-11-15

    The mosquito Aedes aegypti is the vector agent responsible for the transmission of yellow fever and dengue fever viruses to over 80 million people in tropical and subtropical regions of the world. Exhaustive efforts have lead to a vaccine candidate with only 30% effectiveness against the dengue virus and failure to protect patients against the serotype 2. Hence, vector control remains the most viable route to dengue fever control programs. We have synthesized a class of 1,2,4-oxadiazole derivatives whose most biologically active compounds exhibit potent activity against Aedes aegypti larvae (ca. of 15 ppm) and low toxicity in mammals. Exposure to these larvicides results in larvae pigmentation in a manner correlated with the LC50 measurements. Structural comparisons of the 1,2,4-oxadiazole nucleus against known inhibitors of insect enzymes allowed the identification of 3-hydroxykynurenine transaminase as a potential target for these synthetic larvicides. Molecular docking calculations indicate that 1,2,4-oxadiazole compounds can bind to 3-hydroxykynurenine transaminase with similar conformation and binding energies as its crystallographic inhibitor 4-(2-aminophenyl)-4-oxobutanoic acid.

  8. An insecticide resistance-breaking mosquitocide targeting inward rectifier potassium channels in vectors of Zika virus and malaria

    PubMed Central

    Swale, Daniel R.; Engers, Darren W.; Bollinger, Sean R.; Gross, Aaron; Inocente, Edna Alfaro; Days, Emily; Kanga, Fariba; Johnson, Reed M.; Yang, Liu; Bloomquist, Jeffrey R.; Hopkins, Corey R.; Piermarini, Peter M.; Denton, Jerod S.

    2016-01-01

    Insecticide resistance is a growing threat to mosquito control programs around the world, thus creating the need to discover novel target sites and target-specific compounds for insecticide development. Emerging evidence suggests that mosquito inward rectifier potassium (Kir) channels represent viable molecular targets for developing insecticides with new mechanisms of action. Here we describe the discovery and characterization of VU041, a submicromolar-affinity inhibitor of Anopheles (An.) gambiae and Aedes (Ae.) aegypti Kir1 channels that incapacitates adult female mosquitoes from representative insecticide-susceptible and -resistant strains of An. gambiae (G3 and Akron, respectively) and Ae. aegypti (Liverpool and Puerto Rico, respectively) following topical application. VU041 is selective for mosquito Kir channels over several mammalian orthologs, with the exception of Kir2.1, and is not lethal to honey bees. Medicinal chemistry was used to develop an analog, termed VU730, which retains activity toward mosquito Kir1 but is not active against Kir2.1 or other mammalian Kir channels. Thus, VU041 and VU730 are promising chemical scaffolds for developing new classes of insecticides to combat insecticide-resistant mosquitoes and the transmission of mosquito-borne diseases, such as Zika virus, without harmful effects on humans and beneficial insects. PMID:27849039

  9. An insecticide resistance-breaking mosquitocide targeting inward rectifier potassium channels in vectors of Zika virus and malaria.

    PubMed

    Swale, Daniel R; Engers, Darren W; Bollinger, Sean R; Gross, Aaron; Inocente, Edna Alfaro; Days, Emily; Kanga, Fariba; Johnson, Reed M; Yang, Liu; Bloomquist, Jeffrey R; Hopkins, Corey R; Piermarini, Peter M; Denton, Jerod S

    2016-11-16

    Insecticide resistance is a growing threat to mosquito control programs around the world, thus creating the need to discover novel target sites and target-specific compounds for insecticide development. Emerging evidence suggests that mosquito inward rectifier potassium (Kir) channels represent viable molecular targets for developing insecticides with new mechanisms of action. Here we describe the discovery and characterization of VU041, a submicromolar-affinity inhibitor of Anopheles (An.) gambiae and Aedes (Ae.) aegypti Kir1 channels that incapacitates adult female mosquitoes from representative insecticide-susceptible and -resistant strains of An. gambiae (G3 and Akron, respectively) and Ae. aegypti (Liverpool and Puerto Rico, respectively) following topical application. VU041 is selective for mosquito Kir channels over several mammalian orthologs, with the exception of Kir2.1, and is not lethal to honey bees. Medicinal chemistry was used to develop an analog, termed VU730, which retains activity toward mosquito Kir1 but is not active against Kir2.1 or other mammalian Kir channels. Thus, VU041 and VU730 are promising chemical scaffolds for developing new classes of insecticides to combat insecticide-resistant mosquitoes and the transmission of mosquito-borne diseases, such as Zika virus, without harmful effects on humans and beneficial insects.

  10. A potyvirus vector efficiently targets recombinant proteins to chloroplasts, mitochondria and nuclei in plant cells when expressed at the amino terminus of the polyprotein.

    PubMed

    Majer, Eszter; Navarro, José-Antonio; Daròs, José-Antonio

    2015-09-01

    Plant virus-based expression systems allow quick and efficient production of recombinant proteins in plant biofactories. Among them, a system derived from tobacco etch virus (TEV; genus potyvirus) permits coexpression of equimolar amounts of several recombinant proteins. This work analyzed how to target recombinant proteins to different subcellular localizations in the plant cell using this system. We constructed TEV clones in which green fluorescent protein (GFP), with a chloroplast transit peptide (cTP), a nuclear localization signal (NLS) or a mitochondrial targeting peptide (mTP) was expressed either as the most amino-terminal product or embedded in the viral polyprotein. Results showed that cTP and mTP mediated efficient translocation of GFP to the corresponding organelle only when present at the amino terminus of the viral polyprotein. In contrast, the NLS worked efficiently at both positions. Viruses expressing GFP in the amino terminus of the viral polyprotein produced milder symptoms. Untagged GFPs and cTP and NLS tagged amino-terminal GFPs accumulated to higher amounts in infected tissues. Finally, viral progeny from clones with internal GFPs maintained the extra gene better. These observations will help in the design of potyvirus-based vectors able to coexpress several proteins while targeting different subcellular localizations, as required in plant metabolic engineering. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. Self-assembled BolA-like amphiphilic peptides as viral-mimetic gene vectors for cancer cell targeted gene delivery.

    PubMed

    Chen, Jing-Xiao; Xu, Xiao-Ding; Yang, Shuo; Yang, Juan; Zhuo, Ren-Xi; Zhang, Xian-Zheng

    2013-01-01

    In this study, two types of BolA-like amphiphilic peptides with dual ligands comprising a tumor-targeting moiety of RGD sequence and a cell-penetrating moiety of R8 sequence are designed and synthesized as gene vectors. The BolA-structural peptide carriers can self-assemble into spherical nanoparticles with a hydrophilic core and shell, which are similar to the viral capsid and can bind plasmid DNA in an aqueous medium to form viral-mimetic complexes. It is found that the BolA-like dual ligands system exhibits significantly enhanced gene expression in both HeLa and 293T cell lines, as compared with poly(ethylenimine) PEI. These BolA-like amphiphilic peptides are promising in clinical trials of gene therapy. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. A potential peptide vector that allows targeted delivery of a desired fusion protein into the human breast cancer cell line MDA-MB-231.

    PubMed

    Liu, Wei Qing; Yang, Jun; Hong, Min; Gao, Chang E; Dong, Jian

    2016-06-01

    Effective control of breast cancer has been primarily hampered by a lack of tumor specificity in treatments. One potential way to improve targeting specificity is to develop novel vectors that specifically bind to and are internalized by tumor cells. Through a phage display library, an 11-L-amino acid peptide, PI (sequence, CASPSGALRSC), was selected. PI was labeled with fluorescein isothiocyanate (FITC) and named PI-FITC. Subsequently, the specific affinity of PI-FITC to MDA-MB-231 human breast cancer cells and other cancer cell lines was observed by confocal microscopy. Our previous study established that PI-FITC also shows affinity to Calu-1 human lung carcinoma cells and major histocompatibility complex class I antigen molecules; therefore, the cytomembrane proteins of the cell lines were analyzed to determine those that were common to the two cell lines and may be associated with transmembrane transduction. To further test the delivery ability of PI to MDA-MB-231 cells, PI-glutathione-S-transferase (GST) was constructed and the internalization of this fusion protein was visualized by immunofluorescence microscopy. The results revealed that PI exhibited specific affinity to MDA-MB-231 cells. Use of membrane transport inhibitors indicated that macropinocytosis and caveolin-mediated endocytosis may be involved in the endocytosis of PI. In addition, 11 membrane proteins common to MDA-MB-231 and Calu-1 may be associated with transmembrane transduction. In summary, PI was able to deliver PI-GST into MDA-MB-231 cells. Thus, PI could be modified to be a potential vector, and may contribute to the development of targeted therapeutic strategies for breast cancer.

  13. Ligand efficiency-based support vector regression models for predicting bioactivities of ligands to drug target proteins.

    PubMed

    Sugaya, Nobuyoshi

    2014-10-27

    The concept of ligand efficiency (LE) indices is widely accepted throughout the drug design community and is frequently used in a retrospective manner in the process of drug development. For example, LE indices are used to investigate LE optimization processes of already-approved drugs and to re-evaluate hit compounds obtained from structure-based virtual screening methods and/or high-throughput experimental assays. However, LE indices could also be applied in a prospective manner to explore drug candidates. Here, we describe the construction of machine learning-based regression models in which LE indices are adopted as an end point and show that LE-based regression models can outperform regression models based on pIC50 values. In addition to pIC50 values traditionally used in machine learning studies based on chemogenomics data, three representative LE indices (ligand lipophilicity efficiency (LLE), binding efficiency index (BEI), and surface efficiency index (SEI)) were adopted, then used to create four types of training data. We constructed regression models by applying a support vector regression (SVR) method to the training data. In cross-validation tests of the SVR models, the LE-based SVR models showed higher correlations between the observed and predicted values than the pIC50-based models. Application tests to new data displayed that, generally, the predictive performance of SVR models follows the order SEI > BEI > LLE > pIC50. Close examination of the distributions of the activity values (pIC50, LLE, BEI, and SEI) in the training and validation data implied that the performance order of the SVR models may be ascribed to the much higher diversity of the LE-based training and validation data. In the application tests, the LE-based SVR models can offer better predictive performance of compound-protein pairs with a wider range of ligand potencies than the pIC50-based models. This finding strongly suggests that LE-based SVR models are better than pIC50-based

  14. Viral Vector Production: Adenovirus.

    PubMed

    Kim, Julius W; Morshed, Ramin A; Kane, J Robert; Auffinger, Brenda; Qiao, Jian; Lesniak, Maciej S

    2016-01-01

    Adenoviral vectors have proven to be valuable resources in the development of novel therapies aimed at targeting pathological conditions of the central nervous system, including Alzheimer's disease and neoplastic brain lesions. Not only can some genetically engineered adenoviral vectors achieve remarkably efficient and specific gene delivery to target cells, but they also may act as anticancer agents by selectively replicating within cancer cells.Due to the great interest in using adenoviral vectors for various purposes, the need for a comprehensive protocol for viral vector production is especially apparent. Here, we describe the process of generating an adenoviral vector in its entirety, including the more complex process of adenoviral fiber modification to restrict viral tropism in order to achieve more efficient and specific gene delivery.

  15. Further reduction in adenovirus vector-mediated liver transduction without largely affecting transgene expression in target organ by exploiting microrna-mediated regulation and the Cre-loxP recombination system.

    PubMed

    Bennett, David; Sakurai, Fuminori; Shimizu, Kahori; Matsui, Hayato; Tomita, Kyoko; Suzuki, Takayuki; Katayama, Kazufumi; Kawabata, Kenji; Mizuguchi, Hiroyuki

    2012-12-03

    In order to detarget undesirable transduction in the liver by an adenovirus (Ad) vector, we previously demonstrated that insertion of sequences perfectly complementary to liver-specific miR-122a into the 3'-untranslated region (UTR) of transgene specifically reduced the transgene expression in the liver by approximately 100-fold; however, a certain level of residual transgene expression was still found in the liver. In order to further suppress the hepatic transduction, we developed a two-Ad vector system that uses the microRNA (miRNA)-regulated transgene expression system and the Cre-loxP recombination system, i.e., insertion of miR-122a target sequences and loxP sites into the transgene expression cassette and coadministration of a Cre recombinase-expressing Ad vector. In addition, to maintain as much as possible the transgene expression in the spleen, which is the target organ of this study, spleen-specific miR-142-3p target sequences were inserted into the 3'-UTR of the Cre recombinase gene to suppress Cre recombinase expression in the spleen. The spleen is an attractive target for immunotherapy because the spleen plays important roles in the immune system. Coadministration of Ad vector possessing CMV promoter-driven Cre recombinase expression cassette with miR-142-3p target sequences resulted in a further 24-fold reduction in the hepatic transgene expression by the Ad vector containing miR-122a target sequences and loxP sites, compared with coadministration of control Ad vector. On the other hand, there was no significant reduction of transgene expression in the spleen.

  16. Linking Oviposition Site Choice to Offspring Fitness in Aedes aegypti: Consequences for Targeted Larval Control of Dengue Vectors

    PubMed Central

    Wong, Jacklyn; Morrison, Amy C.; Stoddard, Steven T.; Astete, Helvio; Chu, Yui Yin; Baseer, Imaan; Scott, Thomas W.

    2012-01-01

    Background Current Aedes aegypti larval control methods are often insufficient for preventing dengue epidemics. To improve control efficiency and cost-effectiveness, some advocate eliminating or treating only highly productive containers. The population-level outcome of this strategy, however, will depend on details of Ae. aegypti oviposition behavior. Methodology/Principal Findings We simultaneously monitored female oviposition and juvenile development in 80 experimental containers located across 20 houses in Iquitos, Peru, to test the hypothesis that Ae. aegypti oviposit preferentially in sites with the greatest potential for maximizing offspring fitness. Females consistently laid more eggs in large vs. small containers (β = 9.18, p<0.001), and in unmanaged vs. manually filled containers (β = 5.33, p<0.001). Using microsatellites to track the development of immature Ae. aegypti, we found a negative correlation between oviposition preference and pupation probability (β = −3.37, p<0.001). Body size of emerging adults was also negatively associated with the preferred oviposition site characteristics of large size (females: β = −0.19, p<0.001; males: β = −0.11, p = 0.002) and non-management (females: β = −0.17, p<0.001; males: β = −0.11, p<0.001). Inside a semi-field enclosure, we simulated a container elimination campaign targeting the most productive oviposition sites. Compared to the two post-intervention trials, egg batches were more clumped during the first pre-intervention trial (β = −0.17, P<0.001), but not the second (β = 0.01, p = 0.900). Overall, when preferred containers were unavailable, the probability that any given container received eggs increased (β = 1.36, p<0.001). Conclusions/Significance Ae. aegypti oviposition site choice can contribute to population regulation by limiting the production and size of adults. Targeted larval control strategies may unintentionally lead to

  17. Target-specific support vector machine scoring in structure-based virtual screening: computational validation, in vitro testing in kinases, and effects on lung cancer cell proliferation.

    PubMed

    Li, Liwei; Khanna, May; Jo, Inha; Wang, Fang; Ashpole, Nicole M; Hudmon, Andy; Meroueh, Samy O

    2011-04-25

    We assess the performance of our previously reported structure-based support vector machine target-specific scoring function across 41 targets, 40 among them from the Directory of Useful Decoys (DUD). The area under the curve of receiver operating characteristic plots (ROC-AUC) revealed that scoring with SVM-SP resulted in consistently better enrichment over all target families, outperforming Glide and other scoring functions, most notably among kinases. In addition, SVM-SP performance showed little variation among protein classes, exhibited excellent performance in a test case using a homology model, and in some cases showed high enrichment even with few structures used to train a model. We put SVM-SP to the test by virtual screening 1125 compounds against two kinases, EGFR and CaMKII. Among the top 25 EGFR compounds, three compounds (1-3) inhibited kinase activity in vitro with IC₅₀ of 58, 2, and 10 μM. In cell cultures, compounds 1-3 inhibited nonsmall cell lung carcinoma (H1299) cancer cell proliferation with similar IC₅₀ values for compound 3. For CaMKII, one compound inhibited kinase activity in a dose-dependent manner among 20 tested with an IC₅₀ of 48 μM. These results are encouraging given that our in-house library consists of compounds that emerged from virtual screening of other targets with pockets that are different from typical ATP binding sites found in kinases. In light of the importance of kinases in chemical biology, these findings could have implications in future efforts to identify chemical probes of kinases within the human kinome.

  18. Target-Specific Support Vector Machine Scoring in Structure-Based Virtual Screening: Computational Validation, In Vitro Testing in Kinases, and Effects on Lung Cancer Cell Proliferation

    PubMed Central

    Li, Liwei; Khanna, May; Jo, Inha; Wang, Fang; Ashpole, Nicole; Hudmon, Andy; Meroueh, Samy O.

    2011-01-01

    We assess the performance of our previously reported structure-based support vector machine target-specific scoring function across 41 targets, 40 among them from the Directory of Useful Decoys (DUD). The area under the curve of receiver characteristic plots (ROC-AUC) revealed that scoring with SVMSP resulted in consistently better enrichment over all targets families and outperforming Glide and other scoring functions, most notably among kinases. In addition, SVM-SP performance showed little variation among protein classes, exhibited excellent performance in a test case using a homology model, and in some cases showed high enrichment even with few structures used to train a model. We put SVM-SP to the test by virtual screening 1,125 compounds against two kinases, EGFR and CaMKII. Among the top 25 EGFR compounds, three compounds (1–3) inhibited kinase activity in vitro with IC50 of 58, 2, and 10 μM. In cell culture, compounds 1–3 inhibited non-small cell lung carcinoma (H1299) cancer cell proliferation with similar IC50 values for compound 3. For CaMKII, one compound inhibited kinase activity in a dose-dependent manner among 20 tested with an IC50 of 48 μM. These results are encouraging given that our in-house library consists of compounds that emerged from virtual screening of other targets with pockets that are different from typical ATP binding sites found in kinases. In light of the importance of kinases in chemical biology, these findings could have implications in future efforts to identify chemical probes of kinases within the human kinome. PMID:21438548

  19. Hyaluronic acid conjugates as vectors for the active targeting of drugs, genes and nanocomposites in cancer treatment.

    PubMed

    Arpicco, Silvia; Milla, Paola; Stella, Barbara; Dosio, Franco

    2014-03-17

    Hyaluronic acid (HA) is a naturally-occurring glycosaminoglycan and a major component of the extracellular matrix. Low levels of the hyaluronic acid receptor CD44 are found on the surface of epithelial, hematopoietic, and neuronal cells; it is overexpressed in many cancer cells, and in particular in tumor-initiating cells. HA has recently attracted considerable interest in the field of developing drug delivery systems, having been used, as such or encapsulated in different types of nanoassembly, as ligand to prepare nano-platforms for actively targeting drugs, genes, and diagnostic agents. This review describes recent progress made with the several chemical strategies adopted to synthesize conjugates and prepare novel delivery systems with improved behaviors.

  20. Cellular targeting of engineered heterologous antigens is a determinant factor for bovine herpesvirus 4-based vaccine vector development.

    PubMed

    Donofrio, Gaetano; Franceschi, Valentina; Capocefalo, Antonio; Taddei, Simone; Sartori, Chiara; Bonomini, Sabrina; Cavirani, Sandro; Cabassi, Clotilde S; Flammini, Cesidio F

    2009-11-01

    In a previous study, an apathogenic strain of bovine herpesvirus 4 (BoHV-4) cloned as a bacterial artificial chromosome and expressing a chimeric peptide (gE2/gD) as a secreted form was described. Recombinant virus-inoculated animals produced antibodies against bovine viral diarrhea virus (BVDV) gE2 and BoHV-1 gD. However, neutralizing antibodies were produced only against BVDV, not against BoHV-1. In the present work a recombinant BoHV-4 expressing a membrane-linked form of gE2/gD chimeric peptide was constructed, and inoculated rabbits produced serum-neutralizing antibodies against both BVDV and BoHV-1. Protein cell sorting and targeting are a very important issue when immunodominant antigens are engineered for recombinant virus vaccine development.

  1. Targeted suicide gene therapy for glioma using human embryonic stem cell-derived neural stem cells genetically modified by baculoviral vectors.

    PubMed

    Zhao, Y; Lam, D H; Yang, J; Lin, J; Tham, C K; Ng, W H; Wang, S

    2012-02-01

    Tumor-tropic neural stem cells (NSCs) can be used in the Trojan horse approach as cellular vehicles for targeted delivery of therapeutic agents to distant tumor sites. To realize this cancer therapy potential, it is important to have a renewable source to generate large quantities of uniform human NSCs. Here, we reported that NSCs derived from HES1 human embryonic stem cell line were capable of migrating into intracranial glioma xenografts after systemic injection or after intracranial injection at a site distant from the tumor. To test whether the HES1-derived NSCs can be used for cancer gene therapy, we used a baculoviral vector to introduce the herpes simplex virus thymidine kinase suicide gene into the cells and demonstrated that baculovirus-mediated transgene expression may last for at least 3 weeks in NSCs. After being injected into the cerebral hemisphere opposite the tumor site and in the presence of ganciclovir, NSCs expressing the suicide gene were able to inhibit the growth of human glioma xenografts and prolong survival of tumor-bearing mice. Our findings suggest that human embryonic stem cells could potentially serve as a clinically viable source for production of cellular vehicles suitable for targeted anticancer gene therapy.

  2. SU-E-J-115: Correlation of Displacement Vector Fields Calculated by Deformable Image Registration Algorithms with Motion Parameters of CT Images with Well-Defined Targets and Controlled-Motion

    SciTech Connect

    Jaskowiak, J; Ahmad, S; Ali, I; Alsbou, N

    2015-06-15

    Purpose: To investigate correlation of displacement vector fields (DVF) calculated by deformable image registration algorithms with motion parameters in helical axial and cone-beam CT images with motion artifacts. Methods: A mobile thorax phantom with well-known targets with different sizes that were made from water-equivalent material and inserted in foam to simulate lung lesions. The thorax phantom was imaged with helical, axial and cone-beam CT. The phantom was moved with a cyclic motion with different motion amplitudes and frequencies along the superior-inferior direction. Different deformable image registration algorithms including demons, fast demons, Horn-Shunck and iterative-optical-flow from the DIRART software were used to deform CT images for the phantom with different motion patterns. The CT images of the mobile phantom were deformed to CT images of the stationary phantom. Results: The values of displacement vectors calculated by deformable image registration algorithm correlated strongly with motion amplitude where large displacement vectors were calculated for CT images with large motion amplitudes. For example, the maximal displacement vectors were nearly equal to the motion amplitudes (5mm, 10mm or 20mm) at interfaces between the mobile targets lung tissue, while the minimal displacement vectors were nearly equal to negative the motion amplitudes. The maximal and minimal displacement vectors matched with edges of the blurred targets along the Z-axis (motion-direction), while DVF’s were small in the other directions. This indicates that the blurred edges by phantom motion were shifted largely to match with the actual target edge. These shifts were nearly equal to the motion amplitude. Conclusions: The DVF from deformable-image registration algorithms correlated well with motion amplitude of well-defined mobile targets. This can be used to extract motion parameters such as amplitude. However, as motion amplitudes increased, image artifacts increased

  3. Carbon and silver nanoparticles in the fight against the filariasis vector Culex quinquefasciatus: genotoxicity and impact on behavioral traits of non-target aquatic organisms.

    PubMed

    Murugan, Kadarkarai; Nataraj, Devaraj; Madhiyazhagan, Pari; Sujitha, Vasu; Chandramohan, Balamurugan; Panneerselvam, Chellasamy; Dinesh, Devakumar; Chandirasekar, Ramachandran; Kovendan, Kalimuthu; Suresh, Udaiyan; Subramaniam, Jayapal; Paulpandi, Manickam; Vadivalagan, Chithravel; Rajaganesh, Rajapandian; Wei, Hui; Syuhei, Ban; Aziz, Al Thabiani; Alsalhi, Mohamad Saleh; Devanesan, Sandhanasamy; Nicoletti, Marcello; Canale, Angelo; Benelli, Giovanni

    2016-03-01

    Mosquito-borne diseases represent a deadly threat for millions of people worldwide. The Culex genus, with special reference to Culex quinquefasciatus, comprises the most common vectors of filariasis across urban and semi-urban areas of Asia. In recent years, important efforts have been conducted to propose green-synthesized nanoparticles as a valuable alternative to synthetic insecticides. However, the mosquitocidal potential of carbon nanoparticles has been scarcely investigated. In this study, the larvicidal and pupicidal activity of carbon nanoparticle (CNP) and silver nanoparticle (AgNP) was tested against Cx. quinquefasciatus. UV-Vis spectrophotometry, Fourier transform infrared (FTIR) spectroscopy, X-ray diffraction (XRD) analysis, scanning electron microscopy (SEM), transmission electron microscopy (TEM), energy-dispersive X-ray (EDX) spectroscopy, and Raman analysis confirmed the rapid and cheap synthesis of carbon and silver nanoparticles. In laboratory assays, LC50 (lethal concentration that kills 50 % of the exposed organisms) values ranged from 8.752 ppm (first-instar larvae) to 18.676 ppm (pupae) for silver nanoparticles and from 6.373 ppm (first-instar larvae) to 14.849 ppm (pupae) for carbon nanoparticles. The predation efficiency of the water bug Lethocerus indicus after a single treatment with low doses of silver and carbon nanoparticles was not reduced. Moderate evidence of genotoxic effects induced by exposure to carbon nanoparticles was found on non-target goldfish, Carassius auratus. Lastly, the plant extract used for silver nanosynthesis was tested for 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) radical scavenging activity. Overall, our results pointed out that AgNP and CNP can be a candidate for effective tools to reduce larval and pupal populations of filariasis vectors, with reduced genotoxicity and impact on behavioral traits of other aquatic organisms sharing the same ecological

  4. An electron fixed target experiment to search for a new vector boson A' decaying to e+e-

    SciTech Connect

    Rouven Essig; Schuster, Philip; Toro, Natalia; Wojtsekhowski, Bogdan

    2011-02-02

    We describe an experiment to search for a new vector boson A' with weak coupling alpha' > 6 x 10–8 α to electrons (α' = e2/4π) in the mass range 65 MeV < mA' < 550 MeV. New vector bosons with such small couplings arise naturally from a small kinetic mixing of the "dark photon" A' with the photon -- one of the very few ways in which new forces can couple to the Standard Model -- and have received considerable attention as an explanation of various dark matter related anomalies. A' bosons are produced by radiation off an electron beam, and could appear as narrow resonances with small production cross-section in the trident e+e- spectrum. We summarize the experimental approach described in a proposal submitted to Jefferson Laboratory's PAC35, PR-10-009. This experiment, the A' Experiment (APEX), uses the electron beam of the Continuous Electron Beam Accelerator Facility at Jefferson Laboratory (CEBAF) at energies of ~1-4 GeV incident on 0.5-10% radiation length Tungsten wire mesh targets, and measures the resulting e+e- pairs to search for the A' using the High Resolution Spectrometer and the septum magnet in Hall A. With a ~1 month run, APEX will achieve very good sensitivity because the statistics of e+e- pairs will be ~10,000 times larger in the explored mass range than any previous search for the A' boson. These statistics and the excellent mass resolution of the spectrometers allow sensitivity to α'/α one to three orders of magnitude below current limits, in a region of parameter space of great theoretical and phenomenological interest. Similar experiments could also be performed at other facilities, such as the Mainz Microtron.

  5. Target-mediated disposition model describing the dynamics of IL12 and IFNγ after administration of a mifepristone-inducible adenoviral vector for IL-12 expression in mice.

    PubMed

    Parra-Guillen, Zinnia Patricia; Janda, Alvaro; Alzuguren, Pilar; Berraondo, Pedro; Hernandez-Alcoceba, Ruben; Troconiz, Iñaki F

    2013-01-01

    Interleukin-12 (IL12) is a cytokine with potential applications in the treatment of cancer given the potent immune response that it triggers, in part due to its ability to stimulate expression of interferon-γ (IFNγ). To avoid the toxicity associated with systemic exposure to IL12, a high-capacity adenoviral vector carrying a liver-specific, mifepristone-inducible IL12 expression system (HC-Ad/RUmIL12) has been developed. However, the maintenance of IL12 expression at therapeutic levels is compromised by the inhibitory effect of IFNγ on inducible systems. The aim of this work is to develop a semi-mechanistic model to characterize the relationship between IL12 and IFNγ in wild-type and knock-out mice for the IFNγ receptor treated with HC-Ad/RUmIL12 under different dosing regimens in order to better understand the key mechanisms controlling the system. Rapid binding was considered to account for target-mediated disposition exhibited by both cytokines (equilibrium dissociation constant were 18 and 2.28 pM for IL12 and IFNγ, respectively). The final model included: (1) IFNγ receptor turnover, (2) irreversible free cytokine elimination from the serum compartment, (3) internalization of the IL12 receptor complex, (4) IL12 expression upregulated by the co-administration of the adenoviral vector and mifepristone and downregulated by the IFNγ receptor, and (5) synthesis of IFNγ controlled by the relative increments in the bound IL12. In conclusion, a model simultaneously describing the kinetics of IL12 and IFNγ in the context of gene therapy was developed and validated with additional data. The model was applied to design an experimental dosing protocol intended to maintain sustained therapeutic IL12 levels.

  6. The oncolytic herpes simplex virus vector, G47Δ, effectively targets tamoxifen-resistant breast cancer cells.

    PubMed

    Fan, Jingjing; Jiang, Hua; Cheng, Lin; Liu, Renbin

    2016-03-01

    The aim of the present study was to establish a tamoxifen-resistant cell line (MCF-7/TAM-R) and to investigate the therapeutic effect of G47Δ on this cell line both in vitro and in vivo. In the present study, the MCF-7/TAM-R monoclonal subline was established after exposing MCF-7 cells to tamoxifen for 21 days. Then, it was compared with a wild-type MCF-7 subline (MCF-7W), which was not treated with tamoxifen. Cell proliferation, viability, cell cycle and apoptosis analyses were carried out to examine the characteristics of the MCF-7/TAM-R cells. Both in vitro and in vivo toxicity studies were conducted to investigate the therapeutic effect of G47Δ on the MCF-7/TAM-R cells. Compared to the MCF-7W cells, we found that the MCF-7/TAM-R cells exhibited a higher proliferation ability (P<0.05) and a stronger resistance to the cytotoxic effects induced by 4-hydroxytamoxifen (4-OHT) (P<0.05). G47Δ demonstrated a high cytotoxic effect on both the MCF-7/TAM-R and MCF-7W cell lines. After being infected with G47Δ at an MOI of 0.01, >90% of the MCF-7/TAM-R and MCF-7W cells died on day 5. G47Δ induced cell cycle arrest in the G2/M phase. Furthermore, G47Δ inhibited tumor growth in subcutaneous tumor models of both MCF-7/TAM-R and MCF-7W. Thus, we conclude that G47Δ, a third generation oncolytic herpes simplex virus, is highly sensitive and safe in targeting tamoxifen-resistant breast cancer cells both in vitro and in vivo.

  7. Intravitreal delivery of a novel AAV vector targets ON bipolar cells and restores visual function in a mouse model of complete congenital stationary night blindness.

    PubMed

    Scalabrino, Miranda L; Boye, Sanford L; Fransen, Kathryn M H; Noel, Jennifer M; Dyka, Frank M; Min, Seok Hong; Ruan, Qing; De Leeuw, Charles N; Simpson, Elizabeth M; Gregg, Ronald G; McCall, Maureen A; Peachey, Neal S; Boye, Shannon E

    2015-11-01

    Adeno-associated virus (AAV) effectively targets therapeutic genes to photoreceptors, pigment epithelia, Müller glia and ganglion cells of the retina. To date, no one has shown the ability to correct, with gene replacement, an inherent defect in bipolar cells (BCs), the excitatory interneurons of the retina. Targeting BCs with gene replacement has been difficult primarily due to the relative inaccessibility of BCs to standard AAV vectors. This approach would be useful for restoration of vision in patients with complete congenital stationary night blindness (CSNB1), where signaling through the ON BCs is eliminated due to mutations in their G-protein-coupled cascade genes. For example, the majority of CSNB1 patients carry a mutation in nyctalopin (NYX), which encodes a protein essential for proper localization of the TRPM1 cation channel required for ON BC light-evoked depolarization. As a group, CSNB1 patients have a normal electroretinogram (ERG) a-wave, indicative of photoreceptor function, but lack a b-wave due to defects in ON BC signaling. Despite retinal dysfunction, the retinas of CSNB1 patients do not degenerate. The Nyx(nob) mouse model of CSNB1 faithfully mimics this phenotype. Here, we show that intravitreally injected, rationally designed AAV2(quadY-F+T-V) containing a novel 'Ple155' promoter drives either GFP or YFP_Nyx in postnatal Nyx(nob) mice. In treated Nyx(nob) retina, robust and targeted Nyx transgene expression in ON BCs partially restored the ERG b-wave and, at the cellular level, signaling in ON BCs. Our results support the potential for gene delivery to BCs and gene replacement therapy in human CSNB1.

  8. Self-crosslinkable and intracellularly decrosslinkable biodegradable micellar nanoparticles: A robust, simple and multifunctional nanoplatform for high-efficiency targeted cancer chemotherapy.

    PubMed

    Zou, Yan; Fang, Ya; Meng, Hao; Meng, Fenghua; Deng, Chao; Zhang, Jian; Zhong, Zhiyuan

    2016-12-28

    Nanomedicines based on biodegradable micelles offer a most promising treatment for malignant tumors. Their clinical effectiveness, however, remains to be improved. Here, we report that self-crosslinkable and intracellularly decrosslinkable micellar nanoparticles (SCID-Ms) self-assembled from novel amphiphilic biodegradable poly(ethylene glycol)-b-poly(dithiolane trimethylene carbonate) block copolymer achieve high-efficiency targeted cancer chemotherapy in vivo. Interestingly, doxorubicin (DOX)-loaded SCID-Ms showed favorable features of superb stability, minimal drug leakage, long circulation time, triggered drug release inside the tumor cells, and an unprecedented maximum-tolerated dose (MTD) of over 100mg DOX equiv./kg in mice, which was at least 10 times higher than free drug. The in vivo studies in malignant B16 melanoma-bearing C57BL/6 mice revealed that DOX-SCID-Ms at a dosage of 30mg DOX equiv./kg could effectively suppress tumor growth and prolong mice survival time without causing obvious systemic toxicity. Moreover, DOX-SCID-Ms could be readily decorated with a targeting ligand like cRGD peptide. The biodistribution studies showed that cRGD20/DOX-SCID-Ms had a high tumor accumulation of 6.13% ID/g at 6h post injection, which was ca. 3-fold higher than that for clinically used pegylated liposomal doxorubicin (DOX-LPs). Accordingly, cRGD20/DOX-SCID-Ms exhibited significantly better therapeutic efficacy and lower side effects than DOX-LPs in B16 melanoma-bearing mice. These self-regulating biodegradable micellar nanoparticles offer a robust, multifunctional and viable nanoplatform for targeted cancer chemotherapy.

  9. Lentivirus vectors construction of SiRNA targeting interference GPC3 gene and its biological effects on liver cancer cell lines Huh-7.

    PubMed

    Lei, Chang-Jiang; Yao, Chun; Pan, Qing-Yun; Long, Hao-Cheng; Li, Lei; Zheng, Shu-Ping; Zeng, Cheng; Huang, Jian-Bin

    2014-10-01

    To build GPC3 gene short hairpin interference RNA (shRNA) slow virus vector, observe expression of Huh-7 GPC3 gene in human liver cell line proliferation apoptosis and the effect of GPC3 gene influencing on liver cancer cell growth, and provide theoretical basis for gene therapy of liver cancer. Hepatocellular carcinoma cell line Huh-7 was transfected by a RNA interference technique. GPC3 gene expression in a variety of liver cancer cell lines was detected by fluorescence quantitative PCR. Targeted GPC3 gene sequences of small interfering RNA (siRNA) PGC-shRNA-GPC3 were restructured. Stable expression cell lines of siRNA were screened and established with the help of liposomes (lipofectamine(TM2000)) as carrier transfection of human liver cell lines. In order to validate siRNA interference efficiency, GPC3 siRNA mRNA expression was detected after transfection by using RT-PCR and Western blot. The absorbance value of the cells of blank group, untransfection group and transfection group, the cell cycle and cell apoptosis were calculated, and effects of GPC3 gene on Huh-7 cell proliferation and apoptosis were observed. In the liver cancer cell lines Huh-7, GPC3 gene showed high expression. PGC-shRNA-GPC3 recombinant plasmid was constructed successfully via sequencing validation. Stable recombinant plasmid transfected into liver cancer cell lines Huh-7 can obviously inhibit GPC3 mRNA expression level. The targeted GPC3 siRNA can effectively inhibit the expression of GPC3. Copyright © 2014 Hainan Medical College. Published by Elsevier B.V. All rights reserved.

  10. Participatory mapping of target areas to enable operational larval source management to suppress malaria vector mosquitoes in Dar es Salaam, Tanzania.

    PubMed

    Dongus, Stefan; Nyika, Dickson; Kannady, Khadija; Mtasiwa, Deo; Mshinda, Hassan; Fillinger, Ulrike; Drescher, Axel W; Tanner, Marcel; Castro, Marcia C; Killeen, Gerry F

    2007-09-04

    Half of the population of Africa will soon live in towns and cities where it can be protected from malaria by controlling aquatic stages of mosquitoes. Rigorous but affordable and scaleable methods for mapping and managing mosquito habitats are required to enable effective larval control in urban Africa. A simple community-based mapping procedure that requires no electronic devices in the field was developed to facilitate routine larval surveillance in Dar es Salaam, Tanzania. The mapping procedure included (1) community-based development of sketch maps and (2) verification of sketch maps through technical teams using laminated aerial photographs in the field which were later digitized and analysed using Geographical Information Systems (GIS). Three urban wards of Dar es Salaam were comprehensively mapped, covering an area of 16.8 km2. Over thirty percent of this area were not included in preliminary community-based sketch mapping, mostly because they were areas that do not appear on local government residential lists. The use of aerial photographs and basic GIS allowed rapid identification and inclusion of these key areas, as well as more equal distribution of the workload of malaria control field staff. The procedure developed enables complete coverage of targeted areas with larval control through comprehensive spatial coverage with community-derived sketch maps. The procedure is practical, affordable, and requires minimal technical skills. This approach can be readily integrated into malaria vector control programmes, scaled up to towns and cities all over Tanzania and adapted to urban settings elsewhere in Africa.

  11. Effects of camptothecin on tumor cell proliferation and angiogenesis when coupled to a bombesin analog used as a targeted delivery vector.

    PubMed

    Sun, Li-Chun; Luo, Jing; Mackey, Vienna L; Fuselier, Joseph A; Coy, David H

    2007-03-01

    The camptothecin-bombesin conjugate termed DC-51-43, as a novel targeted drug delivery system, was examined in over 10 human tumor cell lines and shows a potent antiproliferative activity. This conjugate has also demonstrated its antitumor activity in our previous experiments. In our present study, we evaluate this conjugate for its antiangiogenic activity by in-vitro and in-vivo experiments. The camptothecin-bombesin conjugate and free camptothecin show potent in-vitro inhibitory activities of cell adhesion to various extracellular matrix components and integrins alphaVbeta3 and alphaVbeta5, not beta1/alphabeta1. This conjugate displays inhibitory activity to cell migration and invasion at concentrations of 10 micromol/l or above. This conjugate is also effective against in-vitro capillary-like tube formation of endothelial cells (at 40 micromol/l), and in-vivo angiogenesis as seen by blocking the spread of host mice endothelial cells into matrigel plugs. These experimental results support the fact that the camptothecin-bombesin conjugate has therapeutic activities against angiogenesis. By binding to bombesin receptor-expressing sites, this bombesin analog, consisting of 11 amino acids, is potentially a novel delivery vector for nonspecific cytotoxic agents.

  12. Antibody-mediated targeted gene transfer of helper virus-free HSV-1 vectors to rat neocortical neurons that contain either NMDA receptor 2B or 2A subunits.

    PubMed

    Cao, Haiyan; Zhang, Guo-rong; Geller, Alfred I

    2011-09-30

    Because of the numerous types of neurons in the brain, and particularly the forebrain, neuron type-specific expression will benefit many potential applications of direct gene transfer. The two most promising approaches for achieving neuron type-specific expression are targeted gene transfer to a specific type of neuron and using a neuron type-specific promoter. We previously developed antibody-mediated targeted gene transfer with Herpes Simplex Virus (HSV-1) vectors by modifying glycoprotein C (gC) to replace the heparin binding domain, which mediates the initial binding of HSV-1 particles to many cell types, with the Staphylococcus A protein ZZ domain, which binds immunoglobulin (Ig) G. We showed that a chimeric gC-ZZ protein is incorporated into vector particles and binds IgG. As a proof-of-principle for antibody-mediated targeted gene transfer, we isolated complexes of these vector particles and an anti-NMDA NR1 subunit antibody, and demonstrated targeted gene transfer to neocortical cells that contain NR1 subunits. However, because most forebrain neurons contain NR1, we obtained only a modest increase in the specificity of gene transfer, and this targeting specificity is of limited utility for physiological experiments. Here, we report efficient antibody-mediated targeted gene transfer to NMDA NR2B- or NR2A-containing cells in rat postrhinal cortex, and a neuron-specific promoter further restricted recombinant expression to neurons. Of note, because NR2A-containing neurons are relatively rare, these results show that antibody-mediated targeted gene transfer with HSV-1 vectors containing neuron type-specific promoters can restrict recombinant expression to specific types of forebrain neurons of physiological significance.

  13. Autonomous parvovirus vectors.

    PubMed

    Maxwell, Ian H; Terrell, Kristina L; Maxwell, Françoise

    2002-10-01

    Parvoviruses are small, icosahedral viruses (approximately 25 nm) containing a single-strand DNA genome (approximately 5 kb) with hairpin termini. Autonomous parvoviruses (APVs) are found in many species; they do not require a helper virus for replication but they do require proliferating cells (S-phase functions) and, in some cases, tissue-specific factors. APVs can protect animals from spontaneous or experimental tumors, leading to consideration of these viruses, and vectors derived from them, as anticancer agents. Vector development has focused on three rodent APVs that can infect human cells, namely, LuIII, MVM, and H1. LuIII-based vectors with complete replacement of the viral coding sequences can direct transient or persistent expression of transgenes in cell culture. MVM-based and H1-based vectors with substitution of transgenes for the viral capsid sequences retain viral nonstructural (NS) coding sequences and express the NS1 protein. The latter serves to amplify the vector genome in target cells, potentially contributing to antitumor activity. APV vectors have packaging capacity for foreign DNA of approximately 4.8 kb, a limit that probably cannot be exceeded by more than a few percent. LuIII vectors can be pseudotyped with capsid proteins from related APVs, a promising strategy for controlling tissue tropism and circumventing immune responses to repeated administration. Initial success has been achieved in targeting such a pseudotyped vector by genetic modification of the capsid. Subject to advances in production and purification methods, APV vectors have potential as gene transfer agents for experimental and therapeutic use, particularly for cancer therapy. Copyright 2002 Elsevier Science (USA)

  14. A Targeted Mutation within the Feline Leukemia Virus (FeLV) Envelope Protein Immunosuppressive Domain To Improve a Canarypox Virus-Vectored FeLV Vaccine

    PubMed Central

    Schlecht-Louf, Géraldine; Mangeney, Marianne; El-Garch, Hanane; Lacombe, Valérie; Poulet, Hervé

    2014-01-01

    We previously delineated a highly conserved immunosuppressive (IS) domain within murine and primate retroviral envelope proteins that is critical for virus propagation in vivo. The envelope-mediated immunosuppression was assessed by the ability of the proteins, when expressed by allogeneic tumor cells normally rejected by engrafted mice, to allow these cells to escape, at least transiently, immune rejection. Using this approach, we identified key residues whose mutation (i) specifically abolishes immunosuppressive activity without affecting the “mechanical” function of the envelope protein and (ii) significantly enhances humoral and cellular immune responses elicited against the virus. The objective of this work was to study the immunosuppressive activity of the envelope protein (p15E) of feline leukemia virus (FeLV) and evaluate the effect of its abolition on the efficacy of a vaccine against FeLV. Here we demonstrate that the FeLV envelope protein is immunosuppressive in vivo and that this immunosuppressive activity can be “switched off” by targeted mutation of a specific amino acid. As a result of the introduction of the mutated envelope sequence into a previously well characterized canarypox virus-vectored vaccine (ALVAC-FeLV), the frequency of vaccine-induced FeLV-specific gamma interferon (IFN-γ)-producing cells was increased, whereas conversely, the frequency of vaccine-induced FeLV-specific interleukin-10 (IL-10)-producing cells was reduced. This shift in the IFN-γ/IL-10 response was associated with a higher efficacy of ALVAC-FeLV against FeLV infection. This study demonstrates that FeLV p15E is immunosuppressive in vivo, that the immunosuppressive domain of p15E can modulate the FeLV-specific immune response, and that the efficacy of FeLV vaccines can be enhanced by inhibiting the immunosuppressive activity of the IS domain through an appropriate mutation. PMID:24198407

  15. Exclusive and common targets of neostriatofugal projections of rat striosome neurons: a single neuron-tracing study using a viral vector.

    PubMed

    Fujiyama, Fumino; Sohn, Jaerin; Nakano, Takashi; Furuta, Takahiro; Nakamura, Kouichi C; Matsuda, Wakoto; Kaneko, Takeshi

    2011-02-01

    The rat neostriatum has a mosaic organization composed of striosome/patch compartments embedded in a more extensive matrix compartment, which are distinguished from each other by the input-output organization as well as by the expression of many molecular markers. The matrix compartment gives rise to the dual γ-aminobutyric acid (GABA)ergic striatofugal systems, i.e. direct and indirect pathway neurons, whereas the striosome compartment is considered to involve direct pathway neurons alone. Although the whole axonal arborization of matrix striatofugal neurons has been examined in vivo by intracellular staining, that of striosome neurons has never been studied at the single neuron level. In the present study, the axonal arborizations of single striosome projection neurons in rat neostriatum were visualized in their entirety using a viral vector expressing membrane-targeted green fluorescent protein, and compared with that of matrix projection neurons. We found that not only matrix but also striosome compartments contained direct and indirect pathway neurons. Furthermore, only striatonigral neurons in the striosome compartment projected directly to the substantia nigra pars compacta (SNc), although they sent a substantial number of axon collaterals to the globus pallidus, entopeduncular nucleus and/or substantia nigra pars reticulata. These results suggest that striosome neurons play a more important role in the formation of reward-related signals of SNc dopaminergic neurons than do matrix neurons. Together with data from previous studies in the reinforcement learning theory, our results suggest that these direct and indirect striosome-SNc pathways together with nigrostriatal dopaminergic neurons may help striosome neurons to acquire the state-value function.

  16. A targeted mutation within the feline leukemia virus (FeLV) envelope protein immunosuppressive domain to improve a canarypox virus-vectored FeLV vaccine.

    PubMed

    Schlecht-Louf, Géraldine; Mangeney, Marianne; El-Garch, Hanane; Lacombe, Valérie; Poulet, Hervé; Heidmann, Thierry

    2014-01-01

    We previously delineated a highly conserved immunosuppressive (IS) domain within murine and primate retroviral envelope proteins that is critical for virus propagation in vivo. The envelope-mediated immunosuppression was assessed by the ability of the proteins, when expressed by allogeneic tumor cells normally rejected by engrafted mice, to allow these cells to escape, at least transiently, immune rejection. Using this approach, we identified key residues whose mutation (i) specifically abolishes immunosuppressive activity without affecting the "mechanical" function of the envelope protein and (ii) significantly enhances humoral and cellular immune responses elicited against the virus. The objective of this work was to study the immunosuppressive activity of the envelope protein (p15E) of feline leukemia virus (FeLV) and evaluate the effect of its abolition on the efficacy of a vaccine against FeLV. Here we demonstrate that the FeLV envelope protein is immunosuppressive in vivo and that this immunosuppressive activity can be "switched off" by targeted mutation of a specific amino acid. As a result of the introduction of the mutated envelope sequence into a previously well characterized canarypox virus-vectored vaccine (ALVAC-FeLV), the frequency of vaccine-induced FeLV-specific gamma interferon (IFN-γ)-producing cells was increased, whereas conversely, the frequency of vaccine-induced FeLV-specific interleukin-10 (IL-10)-producing cells was reduced. This shift in the IFN-γ/IL-10 response was associated with a higher efficacy of ALVAC-FeLV against FeLV infection. This study demonstrates that FeLV p15E is immunosuppressive in vivo, that the immunosuppressive domain of p15E can modulate the FeLV-specific immune response, and that the efficacy of FeLV vaccines can be enhanced by inhibiting the immunosuppressive activity of the IS domain through an appropriate mutation.

  17. Participatory mapping of target areas to enable operational larval source management to suppress malaria vector mosquitoes in Dar es Salaam, Tanzania

    PubMed Central

    Dongus, Stefan; Nyika, Dickson; Kannady, Khadija; Mtasiwa, Deo; Mshinda, Hassan; Fillinger, Ulrike; Drescher, Axel W; Tanner, Marcel; Castro, Marcia C; Killeen, Gerry F

    2007-01-01

    Background Half of the population of Africa will soon live in towns and cities where it can be protected from malaria by controlling aquatic stages of mosquitoes. Rigorous but affordable and scaleable methods for mapping and managing mosquito habitats are required to enable effective larval control in urban Africa. Methods A simple community-based mapping procedure that requires no electronic devices in the field was developed to facilitate routine larval surveillance in Dar es Salaam, Tanzania. The mapping procedure included (1) community-based development of sketch maps and (2) verification of sketch maps through technical teams using laminated aerial photographs in the field which were later digitized and analysed using Geographical Information Systems (GIS). Results Three urban wards of Dar es Salaam were comprehensively mapped, covering an area of 16.8 km2. Over thirty percent of this area were not included in preliminary community-based sketch mapping, mostly because they were areas that do not appear on local government residential lists. The use of aerial photographs and basic GIS allowed rapid identification and inclusion of these key areas, as well as more equal distribution of the workload of malaria control field staff. Conclusion The procedure developed enables complete coverage of targeted areas with larval control through comprehensive spatial coverage with community-derived sketch maps. The procedure is practical, affordable, and requires minimal technical skills. This approach can be readily integrated into malaria vector control programmes, scaled up to towns and cities all over Tanzania and adapted to urban settings elsewhere in Africa. PMID:17784963

  18. RGD-conjugated Nanoparticles for Targeted Inhibition of Metastasis of Integrin alphavbeta3-overexpressing Breast Cancer Cells

    NASA Astrophysics Data System (ADS)

    Shan, Dan

    The use of actively targeted nanoparticles as a delivery system for both the diagnosis and treatment of cancer has been explored extensively. However, selective tumor accumulation is not guaranteed. The objectives of this thesis were 1) to optimize the nanoparticle surface content of cyclic arginyl-glycyl-aspartic acid (cRGD) decorated solid lipid nanoparticles (RGD-SLN) in targeting alphavbeta3 integrin receptor, and 2) to evaluate the potential of RGD-SLN in inhibition of metastasis. Nanoparticles of cRGD content ranging from 0 - 10% mol were synthesized. They showed enhanced binding for alphavbeta3 integrin receptors and increased cellular uptake in the breast cancer cells. In vitro treatment with RGD-SLNs reduced tumor cell adhesion and invasion. Maximum tumor accumulation was demonstrated in 1% mol of RGD on the nanoparticle surface among all formulations tested in vivo. This work has laid a foundation for further development of anticancer drug-loaded cRGD-nanoparticle formulations useful for the treatment of breast cancer metastasis.

  19. Silencing of hepatitis C virus replication by a non-viral vector based on solid lipid nanoparticles containing a shRNA targeted to the internal ribosome entry site (IRES).

    PubMed

    Torrecilla, Josune; Del Pozo-Rodríguez, Ana; Solinís, María Ángeles; Apaolaza, Paola S; Berzal-Herranz, Beatriz; Romero-López, Cristina; Berzal-Herranz, Alfredo; Rodríguez-Gascón, Alicia

    2016-10-01

    Gene silencing mediated by RNAi has gained increasing interest as an alternative for the treatment of infectious diseases such as refractory hepatitis C virus (HCV) infection. In this work we have designed and evaluated a non-viral vector based on solid lipid nanoparticles (SLN) bearing hyaluronic acid, protamine and a short hairpin RNA (shRNA74) targeted to the Internal Ribosome Entry Site (IRES) of the HCV. The vector was able to inhibit the expression of the HCV IRES in Huh-7 cells, with the inhibition level dependent on the shRNA74 to SLN ratio and on the shRNA74 dose added to the culture cells. The nanocarrier was also able to inhibit the replication in human hepatoma cells supporting a subgenomic HCV replicon (Huh-7 NS3-3'). The vector was quickly and efficiently internalized by the cells, and endocytosis was the most productive uptake mechanism for silencing. Clathrin-mediated endocytosis and to a lesser extent caveolae/lipid raft-mediated endocytosis were identified as endocytic mechanisms involved in the cell uptake. Internalization via the CD44 receptor was also involved, although this entry route seems to be less productive for silencing than endocytosis. The vector did not induce either hemolysis or agglutination of red cells in vitro, which was indicative of good biocompatibility. In summary, we have shown for the first time the ability of a non-viral SLN-based vector to silence a HCV replicon.

  20. A Targeted Mulifunctional Platform for Imaging and Treatment of Breast Cancer and Its Metastases Based on Adenoviral Vectors and Magnetic Nanoparticles

    DTIC Science & Technology

    2008-02-01

    synthesis , and regulation of apoptosis (Bridge et al. 1989; Huang et al. 1989). With regards to E4, viral vectors with modifications other than...one of the advantages of HSV-based oncolytic vectors is the potential use of the antiviral drug acyclovir , should replication become out of...such as mRNA transport and shut-off of host cell protein synthesis (Ring 2002). Another type of CRAds are those with tissue specific promoters to

  1. Cloning vector

    DOEpatents

    Guilfoyle, R.A.; Smith, L.M.

    1994-12-27

    A vector comprising a filamentous phage sequence containing a first copy of filamentous phage gene X and other sequences necessary for the phage to propagate is disclosed. The vector also contains a second copy of filamentous phage gene X downstream from a promoter capable of promoting transcription in a bacterial host. In a preferred form of the present invention, the filamentous phage is M13 and the vector additionally includes a restriction endonuclease site located in such a manner as to substantially inactivate the second gene X when a DNA sequence is inserted into the restriction site. 2 figures.

  2. Cloning vector

    DOEpatents

    Guilfoyle, Richard A.; Smith, Lloyd M.

    1994-01-01

    A vector comprising a filamentous phage sequence containing a first copy of filamentous phage gene X and other sequences necessary for the phage to propagate is disclosed. The vector also contains a second copy of filamentous phage gene X downstream from a promoter capable of promoting transcription in a bacterial host. In a preferred form of the present invention, the filamentous phage is M13 and the vector additionally includes a restriction endonuclease site located in such a manner as to substantially inactivate the second gene X when a DNA sequence is inserted into the restriction site.

  3. Equivalent Vectors

    ERIC Educational Resources Information Center

    Levine, Robert

    2004-01-01

    The cross-product is a mathematical operation that is performed between two 3-dimensional vectors. The result is a vector that is orthogonal or perpendicular to both of them. Learning about this for the first time while taking Calculus-III, the class was taught that if AxB = AxC, it does not necessarily follow that B = C. This seemed baffling. The…

  4. Vector quantization

    NASA Technical Reports Server (NTRS)

    Gray, Robert M.

    1989-01-01

    During the past ten years Vector Quantization (VQ) has developed from a theoretical possibility promised by Shannon's source coding theorems into a powerful and competitive technique for speech and image coding and compression at medium to low bit rates. In this survey, the basic ideas behind the design of vector quantizers are sketched and some comments made on the state-of-the-art and current research efforts.

  5. Ultrasound Molecular Imaging of Tumor Angiogenesis with an Integrin Targeted Microbubble Contrast Agent

    PubMed Central

    Anderson, Christopher R.; Hu, Xiaowen; Tlaxca, Jose; Decleves, Anne-Emilie; Houghtaling, Robert; Sharma, Kumar; Lawrence, Michael; Ferrara, Katherine; Rychak, Joshua J.

    2010-01-01

    Rationale and Objectives Ultrasound molecular imaging is an emerging technique for sensitive detection of intravascular targets. Molecular imaging of angiogenesis has strong potential for both clinical use and as a research tool in tumor biology and the development of anti-angiogenic therapies. Our objective is to develop a robust microbubble (MB) ultrasound contrast agent platform to which targeting ligands can be conjugated by biocompatible, covalent conjugation chemistry, and to develop a pure low mechanical index imaging processing method and corresponding quantifying method. The microbubbles and the imaging methods were evaluated in a mouse model of breast cancer in vivo. Materials and Methods We utilized a cyclic RGD (cRGD) pentapeptide containing a terminal cysteine group conjugated to the surface of MB bearing pyridyldithio-propionate (PDP) for targeting αvβ3 integrins. As negative controls, MB without a ligand or MB bearing a scrambled sequence (cRAD) were prepared. To enable characterization of peptides bound to MB surfaces, the cRGD peptide was labeled with FITC and detected by plate fluorometry, flow cytometry, and fluorescence microscopy. Targeted adhesion of cRGD-MB was demonstrated in an in vitro flow adhesion assay against recombinant murine αvβ3 integrin protein and αvβ3 integrin-expressing endothelial cells (bEnd.3). The specificity of cRGD-MB for αvβ3 integrin was demonstrated by treating bEnd.3 EC with a blocking antibody. A murine model of mammary carcinoma was used to assess targeted adhesion and ultrasound molecular imaging in vivo. The targeted microbubbles were visualized using a low mechanical index contrast imaging pulse sequence, and quantified by intensity normalization and two-dimensional Fourier transform analysis, Results The cRGD ligand concentration on the MB surface was ~8.2 × 106 molecules/MB. At a wall shear stress of 1.0 dynes/cm2, cRGD-MB exhibited 5-fold higher adhesion to immobilized recombinant αvβ3 integrin

  6. Targeted gene transfer into ependymal cells through intraventricular injection of AAV1 vector and long-term enzyme replacement via the CSF.

    PubMed

    Yamazaki, Yoshiyuki; Hirai, Yukihiko; Miyake, Koichi; Shimada, Takashi

    2014-07-01

    Enzyme replacement via the cerebrospinal fluid (CSF) has been shown to ameliorate neurological symptoms in model animals with neuropathic metabolic disorders. Gene therapy via the CSF offers a means to achieve a long-term sustainable supply of therapeutic proteins within the central nervous system (CNS) by setting up a continuous source of transgenic products. In the present study, a serotype 1 adeno-associated virus (AAV1) vector was injected into a lateral cerebral ventricle in adult mice to transduce the gene encoding human lysosomal enzyme arylsulfatase A (hASA) into the cells of the CNS. Widespread transduction and stable expression of hASA in the choroid plexus and ependymal cells was observed throughout the ventricles for more than 1 year after vector injection. Although humoral immunity to hASA developed after 6 weeks, which diminished the hASA levels detected in CSF from AAV1-injected mice, hASA levels in CSF were maintained for at least 12 weeks when the mice were tolerized to hASA prior of vector injection. Our results suggest that the cells lining the ventricles could potentially serve as a biological reservoir for long-term continuous secretion of lysosomal enzymes into the CSF following intracerebroventricular injection of an AAV1 vector.

  7. Innovative technologies targeting vector populations to mitigate the risk of exposure to leishmaniasis and protect deployed U.S. Military personnel in the Middle East

    USDA-ARS?s Scientific Manuscript database

    Phlebotomine sand flies, including Phlebotomus papatasi, are blood feeders and vectors of significant public health importance because they transmit Leishmania spp., which cause leishmaniasis. Deployed U.S. Military personnel in the Middle East suffer from sand fly bites and are at risk of contract...

  8. Innovative technologies targeting vector populations to mitigate the risk of exposure to leishmaniasis and protect deployed U.S. Military personnel in the Middle East

    USDA-ARS?s Scientific Manuscript database

    Phlebotomine sand flies, including Phlebotomus papatasi, are blood feeders and vectors of significant public health importance because they transmit Leishmania spp., which cause leishmaniasis. Deployed U.S. Military personnel in the Middle East suffer from sand fly bites and are at risk of contracti...

  9. Evaluation of attractive toxic sugar bait (ATSB)—barrier for control of vector and nuisance mosquitoes and its effect on non-target organisms in sub-tropical environments in Florida

    PubMed Central

    Qualls, Whitney A.; Müller, Günter C.; Revay, Edita E.; Allan, Sandra A.; Arheart, Kristopher L.; Beier, John C.; Smith, Michal L.; Scott, Jodi M.; Kravchenko, Vasiliy D.; Hausmann, Axel; Yefremova, Zoya A.; Xue, Rui-De

    2014-01-01

    The efficacy of attractive toxic sugar baits (ATSB) with the active ingredient eugenol, an Environmental Protection Agency exempt compound, was evaluated against vector and nuisance mosquitoes in both laboratory and field studies. In the laboratory, eugenol combined in attractive sugar bait (ASB) solution provided high levels of mortality for Aedes aegypti, Culex quinquefasciatus, and Anopheles quadrimaculatus. Field studies demonstrated significant control: > 70% reduction for Aedes atlanticus, Ae. infirmatus, and Culex nigripalpus and > 50% reduction for An. crucians, Uranotaenia sapphirina, Culiseta melanura, and Cx. erraticus three weeks post ATSB application. Furthermore, non-target feeding of six insect orders, Hymenoptera, Lepidoptera, Coleoptera, Diptera, Hemiptera, and Orthoptera, was evaluated in the field after application of a dyed-ASB to flowering and non-flowering vegetation. ASB feeding (staining) was determined by dissecting the guts and searching for food dye with a dissecting microscope. The potential impact of ATSB on non-targets, applied on green non-flowering vegetation was low for all non-target groups (0.9%). However, application of the ASB to flowering vegetation resulted in significant staining of the non-target insect orders. This highlights the need for application guidelines to reduce non-target effects. No mortality was observed in laboratory studies with predatory non-targets, spiders, praying mantis, or ground beetles, after feeding for three days on mosquitoes engorged on ATSB. Overall, our laboratory and field studies support the use of eugenol as an active ingredient for controlling important vector and nuisance mosquitoes when used as an ATSB toxin. This is the first study demonstrating effective control of anophelines in non-arid environments which suggest that even in highly competitive sugar rich environments this method could be used for control of malaria in Latin American countries. PMID:24361724

  10. Evaluation of attractive toxic sugar bait (ATSB)-Barrier for control of vector and nuisance mosquitoes and its effect on non-target organisms in sub-tropical environments in Florida.

    PubMed

    Qualls, Whitney A; Müller, Günter C; Revay, Edita E; Allan, Sandra A; Arheart, Kristopher L; Beier, John C; Smith, Michal L; Scott, Jodi M; Kravchenko, Vasiliy D; Hausmann, Axel; Yefremova, Zoya A; Xue, Rui-De

    2014-03-01

    The efficacy of attractive toxic sugar baits (ATSB) with the active ingredient eugenol, an Environmental Protection Agency exempt compound, was evaluated against vector and nuisance mosquitoes in both laboratory and field studies. In the laboratory, eugenol combined in attractive sugar bait (ASB) solution provided high levels of mortality for Aedes aegypti, Culex quinquefasciatus, and Anopheles quadrimaculatus. Field studies demonstrated significant control: >70% reduction for Aedes atlanticus, Aedes. infirmatus, and Culex nigripalpus and >50% reduction for Anopheles crucians, Uranotaenia sapphirina, Culiseta melanura, and Culex erraticus three weeks post ATSB application. Furthermore, non-target feeding of six insect orders, Hymenoptera, Lepidoptera, Coleoptera, Diptera, Hemiptera, and Orthoptera, was evaluated in the field after application of a dyed-ASB to flowering and non-flowering vegetation. ASB feeding (staining) was determined by dissecting the guts and searching for food dye with a dissecting microscope. The potential impact of ATSB on non-targets, applied on green non-flowering vegetation was low for all non-target groups (0.9%). However, application of the ASB to flowering vegetation resulted in significant staining of the non-target insect orders. This highlights the need for application guidelines to reduce non-target effects. No mortality was observed in laboratory studies with predatory non-targets, spiders, praying mantis, or ground beetles, after feeding for three days on mosquitoes engorged on ATSB. Overall, our laboratory and field studies support the use of eugenol as an active ingredient for controlling important vector and nuisance mosquitoes when used as an ATSB toxin. This is the first study demonstrating effective control of anophelines in non-arid environments which suggest that even in highly competitive sugar rich environments this method could be used for control of malaria in Latin American countries.

  11. Vector carpets

    SciTech Connect

    Dovey, D.

    1995-03-22

    Previous papers have described a general method for visualizing vector fields that involves drawing many small ``glyphs`` to represent the field. This paper shows how to improve the speed of the algorithm by utilizing hardware support for line drawing and extends the technique from regular to unstructured grids. The new approach can be used to visualize vector fields at arbitrary surfaces within regular and unstructured grids. Applications of the algorithm include interactive visualization of transient electromagnetic fields and visualization of velocity fields in fluid flow problems.

  12. Extension of On-Surface Radiation Condition (OSRC) Theory to Full-Vector Electromagnetic Wave Scattering by Three-Dimensional Conducting, Dielectric, and Coated Targets

    DTIC Science & Technology

    1993-08-27

    SECURITY CLASSIFICATION AUTHORITY 3 DISTRIBUTION/AVAILABILITY OF REPORT 2b DECLASSIFICATION i DOWNGRADING SCHEDULE UNLIMITED 4. PERFORMING...NO. 11 TITLE (Include Security Classfication) Extension of OSRC to Full-Vector Electromagnetic Wave Scattering (UNCLASSIFIED) 12 PERSONAL AUTHOR (S... authors wish to thank the referees for their constructive remarks. They also wish to thank Mr. Thomas Moore for his Vii. RELATION TO PREVIOUS HIGH

  13. A Targeted Multifunctional Platform for Imaging and Treatment of Breast Cancer and Its Metastases Based on Adenoviral Vectors and Magnetic Nanoparticles

    DTIC Science & Technology

    2007-08-01

    our main contact person. Dr. Nikles is the Associate Director of the Center for Materials for Information Technology and an expert in the synthesis ...DNA replication, mRNA transport and splicing, in- hibition of host cell protein synthesis , and regulation of apoptosis (Bridge et al. 1989; Huang et al...potential use of the antiviral drug acyclovir , should replication become out of control. HSV-1 based vectors have been tested in various phases of clinical

  14. A novel solid lipid nanoparticle formulation for active targeting to tumor α(v) β(3) integrin receptors reveals cyclic RGD as a double-edged sword.

    PubMed

    Shuhendler, Adam J; Prasad, Preethy; Leung, Michael; Rauth, Andrew M; Dacosta, Ralph S; Wu, Xiao Yu

    2012-09-01

    The overexpression of α(v) β(3) integrin receptors on tumor cells and tumor vascular endothelium makes it a useful target for imaging, chemotherapy and anti-angiogenic therapy. However integrin-targeted delivery of therapeutics by nanoparticles have provided only marginal, if any, enhancement of therapeutic effect. This work was thus focused on the development of novel α(v) β(3) -targeted near infrared light-emitting solid lipid nanoparticles (SLN) through conjugation to the α(v) β(3) integrin-specific ligand cyclic Arg-Gly-Asp (cRGD), and the assessment of the effects of α(v) β(3) targeting on nanoparticle biodistribution. Since our previously developed non-targeted "stealth" SLN showed little hepatic accumulation, unlike most reported liposomes and micelles, they served as a reference for quantifying the effects of cRGD-conjugation on tumor uptake and whole animal biodistribution of SLN. Non-targeted SLN, actively targeted (RGD-SLN) and blocked RGD-SLN were prepared to contain near infrared quantum dots for live animal imaging. They were injected intravenously to nude mice bearing xenograft orthotopic human breast tumors or dorsal window chamber breast tumors. Tumor micropharmacokinetics of various SLN formulations were determined using intravital microscopy, and whole animal biodistribution was followed over time by optical imaging. The active tumor targeting with cRGD was found to be a "double-edged sword": while the specificity of RGD-SLN accumulation in tumor blood vessels and their tumor residence time increased, their distribution in the liver, spleen, and kidneys was significantly greater than the non-targeted SLN, leaving a smaller amount of nanoparticles in the tumor tissue. Nevertheless the enhanced specificity and retention of RGD-SLN in tumor neovasculature could make this novel formulation useful for tumor neovascular-specific therapies and imaging applications. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. Safety considerations in vector development.

    PubMed

    Kappes, J C; Wu, X

    2001-11-01

    The inadvertent production of replication competent retrovirus (RCR) constitutes the principal safety concern for the use of lentiviral vectors in human clinical protocols. Because of limitations in animal models to evaluate lentiviral vectors for their potential to recombine and induce disease, the vector design itself should ensure against the emergence of RCR in vivo. Issues related to RCR generation and one approach to dealing with this problem are discussed in this chapter. To assess the risk of generating RCR, a highly sensitive biological assay was developed to specifically detect vector recombination in transduced cells. Analysis of lentiviral vector stocks has shown that recombination occurs during reverse transcription in primary target cells. Rejoining of viral protein-coding sequences of the packaging construct and cis-acting sequences of the vector was demonstrated to generate env-minus recombinants (LTR-gag-pol-LTR). Mobilization of recombinant lentiviral genomes was also demonstrated but was dependent on pseudotyping of the vector core with an exogenous envelope protein. 5' sequence analysis has demonstrated that recombinants consist of U3, R, U5, and the psi packaging signal joined with an open gag coding region. Analysis of the 3' end has mapped the point of vector recombination to the poly(A) tract of the packaging construct's mRNA. The state-of-the-art third generation packaging construct and SIN vector also have been shown to generate env-minus proviral recombinants capable of mobilizing retroviral DNA when pseudotyped with an exogenous envelope protein. A new class of HIV-based vector (trans-vector) was recently developed that splits the gag-pol component of the packaging construct into two parts: one that expresses Gag/Gag-Pro and another that expresses Pol (RT and IN) fused with Vpr. Unlike other lentiviral vectors, the trans-vector has not been shown to form recombinants capable of DNA mobilization. These results indicate the trans-vector

  16. Designing plasmid vectors.

    PubMed

    Tolmachov, Oleg

    2009-01-01

    Nonviral gene therapy vectors are commonly based on recombinant bacterial plasmids or their derivatives. The plasmids are propagated in bacteria, so, in addition to their therapeutic cargo, they necessarily contain a bacterial replication origin and a selection marker, usually a gene conferring antibiotic resistance. Structural and maintenance plasmid stability in bacteria is required for the plasmid DNA production and can be achieved by carefully choosing a combination of the therapeutic DNA sequences, replication origin, selection marker, and bacterial strain. The use of appropriate promoters, other regulatory elements, and mammalian maintenance devices ensures that the therapeutic gene or genes are adequately expressed in target human cells. Optimal immune response to the plasmid vectors can be modulated via inclusion or exclusion of DNA sequences containing immunostimulatory CpG sequence motifs. DNA fragments facilitating construction of plasmid vectors should also be considered for inclusion in the design of plasmid vectors. Techniques relying on site-specific or homologous recombination are preferred for construction of large plasmids (>15 kb), while digestion of DNA by restriction enzymes with subsequent ligation of the resulting DNA fragments continues to be the mainstream approach for generation of small- and medium-size plasmids. Rapid selection of a desired recombinant plasmid against a background of other plasmids continues to be a challenge. In this chapter, the emphasis is placed on efficient and flexible versions of DNA cloning protocols using selection of recombinant plasmids by restriction endonucleases directly in the ligation mixture.

  17. Identification of the transcriptional regulatory sequences of human calponin promoter and their use in targeting a conditionally replicating herpes vector to malignant human soft tissue and bone tumors.

    PubMed

    Yamamura, H; Hashio, M; Noguchi, M; Sugenoya, Y; Osakada, M; Hirano, N; Sasaki, Y; Yoden, T; Awata, N; Araki, N; Tatsuta, M; Miyatake, S I; Takahashi, K

    2001-05-15

    The calponin (basic or h1) gene, normally expressed in maturated smooth muscle cells, is aberrantly expressed in a variety of human soft tissue and bone tumors. In this study, we show that expression of the calponin gene in human soft tissue and bone tumor cells is regulated at the transcriptional level by the sequence between positions -260 and -219 upstream of the translation initiation site. A novel conditionally replicating herpes simplex virus-1 vector (d12.CALP) in which the calponin promoter drives expression of ICP4, a major trans-activating factor for viral genes was constructed and tested as an experimental treatment for malignant human soft tissue and bone tumors. In cell culture, d12.CALP at low multiplicity of infection (0.001 plaque-forming unit/cell) selectively killed calponin-positive human synovial sarcoma, leiomyosarcoma, and osteosarcoma cells. For in vivo studies, 10 animals harboring SK-LMS-1 human leiomyosarcoma cells were randomly divided and treated twice on days 0 and 9 intraneoplastically with either 1 x 10(7) plaque-forming units of d12.CALP/100 mm(3) of tumor volume or with medium alone. The viral treatment group showed stable and significant inhibition of tumorigenicity with apparent cure in four of five mice by day 35. Replication of viral DNA demonstrated by PCR amplification and expression of the inserted LacZ gene visualized by 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside histochemistry was associated with oncolysis of d12.CALP-treated tumors, while sparing normal vascular smooth muscle cells. In mice harboring two SK-LMS-1 tumors, replication of d12.CALP was detected in a nontreated tumor distant from the site of virus inoculation. These results indicate that replication-competent virus vectors controlled by the calponin transcriptional regulatory sequence may be a new therapeutic strategy for treatment of malignant human soft tissue and bone tumors.

  18. Multiple Insecticide Resistance in the Malaria Vector Anopheles funestus from Northern Cameroon Is Mediated by Metabolic Resistance Alongside Potential Target Site Insensitivity Mutations

    PubMed Central

    Menze, Benjamin D.; Riveron, Jacob M.; Ibrahim, Sulaiman S.; Irving, Helen; Antonio-Nkondjio, Christophe; Awono-Ambene, Parfait H.; Wondji, Charles S.

    2016-01-01

    Background Despite the recent progress in establishing the patterns of insecticide resistance in the major malaria vector Anopheles funestus, Central African populations of this species remain largely uncharacterised. To bridge this important gap and facilitate the implementation of suitable control strategies against this vector, we characterised the resistance patterns of An. funestus population from northern Cameroon. Methods and Findings Collection of indoor-resting female mosquitoes in Gounougou (northern Cameroon) in 2012 and 2015 revealed a predominance of An. funestus during dry season. WHO bioassays performed using F1 An. funestus revealed that the population was multiple resistant to several insecticide classes including pyrethroids (permethrin, deltamethrin, lambda-cyhalothrin and etofenprox), carbamates (bendiocarb) and organochlorines (DDT and dieldrin). However, a full susceptibility was observed against the organophosphate malathion. Bioassays performed with 2015 collection revealed that resistance against pyrethroids and DDT is increasing. PBO synergist assays revealed a significant recovery of susceptibility for all pyrethroids but less for DDT. Analysis of the polymorphism of a portion of the voltage-gated sodium channel gene (VGSC) revealed the absence of the L1014F/S kdr mutation but identified 3 novel amino acid changes I877L, V881L and A1007S. However, no association was established between VGSC polymorphism and pyrethroid/DDT resistance. The DDT resistant 119F-GSTe2 allele (52%) and the dieldrin resistant 296S-RDL allele (45%) were detected in Gounougou. Temporal analysis between 2006, 2012 and 2015 collections revealed that the 119F-GSTe2 allele was relatively stable whereas a significant decrease is observed for 296S-RDL allele. Conclusion This multiple resistance coupled with the temporal increased in resistance intensity highlights the need to take urgent measures to prolong the efficacy of current insecticide-based interventions against

  19. Oviposition by African malaria vector mosquitoes. II. Effects of site tone, water type and conspecific immatures on target selection by freshwater Anopheles gambiae Giles, sensu lato.

    PubMed

    McCrae, A W

    1984-06-01

    Females of Anopheles gambiae s. lat., most of which would have been A. gambiae s. str., were collected from houses in coastal Kenya and tested for their oviposition preferences using Petri dishes in large laboratory cages with lighting equivalent to weak moonlight. Significantly more eggs were laid overnight in water over black than over paler tones, and this difference increased as contrast with the surrounding floor was increased. Direct observation revealed that over white targets, females oviposited from a settled posture, whereas over black targets they did so from flight. The influence on this behaviour of target darkness (tone) overrode that of cage size or target size. In tests which yielded markedly fewer eggs in sea water than in tap water, no significant difference was detected when cage floors were either black or white, although a black floor might have resulted in significantly greater discrimination against sea water had more tests been conducted. All further testing was done over black cage floors. Turbid water from a natural development site received more eggs than distilled, tap or swamp water, even though the turbid water appeared paler than the others. The females did not discriminate between rearing water and tap water, or tap water with and without pupae, but the presence of larvae was repellent. Turbid water from a development site thus seemed to possess an arrestant property which overrode selection favouring darker targets, and which was not derived from prior presence of conspecific immatures. It is suggested that for A. gambiae, oviposition from a settled posture is a response to sub-optimal stimuli, possibly indicating conditions under which oviposition would not occur in nature, and hence why cage experiments using white targets have in the past yielded confusing results.

  20. Multimodality Imaging with Silica-Based Targeted Nanoparticle Platforms

    SciTech Connect

    Jason S. Lewis

    2012-04-09

    Objectives: To synthesize and characterize a C-Dot silica-based nanoparticle containing 'clickable' groups for the subsequent attachment of targeting moieties (e.g., peptides) and multiple contrast agents (e.g., radionuclides with high specific activity) [1,2]. These new constructs will be tested in suitable tumor models in vitro and in vivo to ensure maintenance of target-specificity and high specific activity. Methods: Cy5 dye molecules are cross-linked to a silica precursor which is reacted to form a dye-rich core particle. This core is then encapsulated in a layer of pure silica to create the core-shell C-Dot (Figure 1) [2]. A 'click' chemistry approach has been used to functionalize the silica shell with radionuclides conferring high contrast and specific activity (e.g. 64Cu and 89Zr) and peptides for tumor targeting (e.g. cRGD and octreotate) [3]. Based on the selective Diels-Alder reaction between tetrazine and norbornene, the reaction is bioorthogonal, highyielding, rapid, and water-compatible. This radiolabeling approach has already been employed successfully with both short peptides (e.g. octreotate) and antibodies (e.g. trastuzumab) as model systems for the ultimate labeling of the nanoparticles [1]. Results: PEGylated C-Dots with a Cy5 core and labeled with tetrazine have been synthesized (d = 55 nm, zeta potential = -3 mV) reliably and reproducibly and have been shown to be stable under physiological conditions for up to 1 month. Characterization of the nanoparticles revealed that the immobilized Cy5 dye within the C-Dots exhibited fluorescence intensities over twice that of the fluorophore alone. The nanoparticles were successfully radiolabeled with Cu-64. Efforts toward the conjugation of targeting peptides (e.g. cRGD) are underway. In vitro stability, specificity, and uptake studies as well as in vivo imaging and biodistribution investigations will be presented. Conclusions: C-Dot silica-based nanoparticles offer a robust, versatile, and multi

  1. Gene Therapy for Neuropathic Pain by Silencing of TNF-α Expression with Lentiviral Vectors Targeting the Dorsal Root Ganglion in Mice

    PubMed Central

    Ogawa, Nobuhiro; Kawai, Hiromichi; Terashima, Tomoya; Kojima, Hideto; Oka, Kazuhiro; Chan, Lawrence; Maegawa, Hiroshi

    2014-01-01

    Neuropathic pain can be a debilitating condition. Many types of drugs that have been used to treat neuropathic pain have only limited efficacy. Recent studies indicate that pro-inflammatory mediators including tumor necrosis factor α (TNF-α) are involved in the pathogenesis of neuropathic pain. In the present study, we engineered a gene therapy strategy to relieve neuropathic pain by silencing TNF-α expression in the dorsal root ganglion (DRG) using lentiviral vectors expressing TNF short hairpin RNA1-4 (LV-TNF-shRNA1-4) in mice. First, based on its efficacy in silencing TNF-α in vitro, we selected shRNA3 to construct LV-TNF-shRNA3 for in vivo study. We used L5 spinal nerve transection (SNT) mice as a neuropathic pain model. These animals were found to display up-regulated mRNA expression of activating transcription factor 3 (ATF3) and neuropeptide Y (NPY), injury markers, and interleukin (IL)-6, an inflammatory cytokine in the ipsilateral L5 DRG. Injection of LV-TNF-shRNA3 onto the proximal transected site suppressed significantly the mRNA levels of ATF3, NPY and IL-6, reduced mechanical allodynia and neuronal cell death of DRG neurons. These results suggest that lentiviral-mediated silencing of TNF-α in DRG relieves neuropathic pain and reduces neuronal cell death, and may constitute a novel therapeutic option for neuropathic pain. PMID:24642694

  2. Does Cattle Milieu Provide a Potential Point to Target Wild Exophilic Anopheles arabiensis (Diptera: Culicidae) with Entomopathogenic Fungus? A Bioinsecticide Zooprophylaxis Strategy for Vector Control

    PubMed Central

    Lyimo, Issa N.; Ng'habi, Kija R.; Mpingwa, Monica W.; Daraja, Ally A.; Mwasheshe, Dickson D.; Nchimbi, Nuru S.; Lwetoijera, Dickson W.; Mnyone, Ladslaus L.

    2012-01-01

    Background. Anopheles arabiensis is increasingly dominating malaria transmission in Africa. The exophagy in mosquitoes threatens the effectiveness of indoor vector control strategies. This study aimed to evaluate the effectiveness of fungus against An. arabiensis when applied on cattle and their environments. Methods. Experiments were conducted under semi-field and small-scale field conditions within Kilombero valley. The semi-field reared females of 5–7 days old An. arabiensis were exposed to fungus-treated and untreated calf. Further, wild An. arabiensis were exposed to fungus-treated calves, mud-huts, and their controls. Mosquitoes were recaptured the next morning and proportion fed, infected, and survived were evaluated. Experiments were replicated three times using different individuals of calves. Results. A high proportion of An. arabiensis was fed on calves (>0.90) and become infected (0.94) while resting on fungus-treated mud walls than on other surfaces. However, fungus treatments reduced fecundity and survival of mosquitoes. Conclusion. This study demonstrates for the first time the potential of cattle and their milieu for controlling An. arabiensis. Most of An. arabiensis were fed and infected while resting on fungus-treated mud walls than on other surfaces. Fungus treatments reduced fecundity and survival of mosquitoes. These results suggest deployment of bioinsecticide zooprophylaxis against exophilic An. arabiensis. PMID:22934152

  3. LV305, a dendritic cell-targeting integration-deficient ZVex(TM)-based lentiviral vector encoding NY-ESO-1, induces potent anti-tumor immune response.

    PubMed

    Albershardt, Tina Chang; Campbell, David James; Parsons, Andrea Jean; Slough, Megan Merrill; Ter Meulen, Jan; Berglund, Peter

    2016-01-01

    We have engineered an integration-deficient lentiviral vector, LV305, to deliver the tumor antigen NY-ESO-1 to human dendritic cells in vivo through pseudotyping with a modified Sindbis virus envelop protein. Mice immunized once with LV305 developed strong, dose-dependent, multifunctional, and cytotoxic NY-ESO-1-specific cluster of differentiation 8 (CD8) T cells within 14 days post-immunization and could be boosted with LV305 at least twice to recall peak-level CD8 T-cell responses. Immunization with LV305 protected mice against tumor growth in an NY-ESO-1-expressing CT26 lung metastasis model, with the protective effect abrogated upon depletion of CD8 T cells. Adoptive transfer of CD8 T cells, alone or together with CD4 T cells or natural killer cells, from LV305-immunized donor mice to tumor-bearing recipient mice conferred significant protection against metastatic tumor growth. Biodistribution of injected LV305 in mice was limited to the site of injection and the draining lymph node, and injected LV305 exhibited minimal excretion. Mice injected with LV305 developed little to no adverse effects, as evaluated by toxicology studies adherent to good laboratory practices. Taken together, these data support the development of LV305 as a clinical candidate for treatment against tumors expressing NY-ESO-1.

  4. LV305, a dendritic cell-targeting integration-deficient ZVexTM-based lentiviral vector encoding NY-ESO-1, induces potent anti-tumor immune response

    PubMed Central

    Albershardt, Tina Chang; Campbell, David James; Parsons, Andrea Jean; Slough, Megan Merrill; ter Meulen, Jan; Berglund, Peter

    2016-01-01

    We have engineered an integration-deficient lentiviral vector, LV305, to deliver the tumor antigen NY-ESO-1 to human dendritic cells in vivo through pseudotyping with a modified Sindbis virus envelop protein. Mice immunized once with LV305 developed strong, dose-dependent, multifunctional, and cytotoxic NY-ESO-1-specific cluster of differentiation 8 (CD8) T cells within 14 days post-immunization and could be boosted with LV305 at least twice to recall peak-level CD8 T-cell responses. Immunization with LV305 protected mice against tumor growth in an NY-ESO-1-expressing CT26 lung metastasis model, with the protective effect abrogated upon depletion of CD8 T cells. Adoptive transfer of CD8 T cells, alone or together with CD4 T cells or natural killer cells, from LV305-immunized donor mice to tumor-bearing recipient mice conferred significant protection against metastatic tumor growth. Biodistribution of injected LV305 in mice was limited to the site of injection and the draining lymph node, and injected LV305 exhibited minimal excretion. Mice injected with LV305 developed little to no adverse effects, as evaluated by toxicology studies adherent to good laboratory practices. Taken together, these data support the development of LV305 as a clinical candidate for treatment against tumors expressing NY-ESO-1. PMID:27626061

  5. Does Cattle Milieu Provide a Potential Point to Target Wild Exophilic Anopheles arabiensis (Diptera: Culicidae) with Entomopathogenic Fungus? A Bioinsecticide Zooprophylaxis Strategy for Vector Control.

    PubMed

    Lyimo, Issa N; Ng'habi, Kija R; Mpingwa, Monica W; Daraja, Ally A; Mwasheshe, Dickson D; Nchimbi, Nuru S; Lwetoijera, Dickson W; Mnyone, Ladslaus L

    2012-01-01

    Background. Anopheles arabiensis is increasingly dominating malaria transmission in Africa. The exophagy in mosquitoes threatens the effectiveness of indoor vector control strategies. This study aimed to evaluate the effectiveness of fungus against An. arabiensis when applied on cattle and their environments. Methods. Experiments were conducted under semi-field and small-scale field conditions within Kilombero valley. The semi-field reared females of 5-7 days old An. arabiensis were exposed to fungus-treated and untreated calf. Further, wild An. arabiensis were exposed to fungus-treated calves, mud-huts, and their controls. Mosquitoes were recaptured the next morning and proportion fed, infected, and survived were evaluated. Experiments were replicated three times using different individuals of calves. Results. A high proportion of An. arabiensis was fed on calves (>0.90) and become infected (0.94) while resting on fungus-treated mud walls than on other surfaces. However, fungus treatments reduced fecundity and survival of mosquitoes. Conclusion. This study demonstrates for the first time the potential of cattle and their milieu for controlling An. arabiensis. Most of An. arabiensis were fed and infected while resting on fungus-treated mud walls than on other surfaces. Fungus treatments reduced fecundity and survival of mosquitoes. These results suggest deployment of bioinsecticide zooprophylaxis against exophilic An. arabiensis.

  6. Viral Vectors: The Road to Reducing Genotoxicity.

    PubMed

    David, Rhiannon M; Doherty, Ann T

    2017-02-01

    Viral vector use in gene therapy has highlighted several safety concerns, including genotoxic events. Generally, vector-mediated genotoxicity results from upregulation of cellular proto-oncogenes via promoter insertion, promoter activation, or gene transcript truncation, with enhancer-mediated activation of nearby genes the primary mechanism reported in gene therapy trials. Vector-mediated genotoxicity can be influenced by virus type, integration target site, and target cell type; different vectors have distinct integration profiles which are cell-specific. Non-viral factors, including patient age, disease, and dose can also influence genotoxic potential, thus the choice of test models and clinical trial populations is important to ensure they are indicative of efficacy and safety. Efforts have been made to develop viral vectors with less risk of insertional mutagenesis, including self-inactivating (SIN) vectors, enhancer-blocking insulators, and microRNA targeting of vectors, although insertional mutagenesis is not completely abrogated. Here we provide an overview of the current understanding of viral vector-mediated genotoxicity risk from factors contributing to viral vector-mediated genotoxicity to efforts made to reduce genotoxicity, and testing strategies required to adequately assess the risk of insertional mutagenesis. It is clear that there is not a 'one size fits all' approach to vector modification for reducing genotoxicity, and addressing these challenges will be a key step in the development of therapies such as CRISPR-Cas9 and delivery of future gene-editing technologies.

  7. Targeting different types of human meningioma and glioma cells using a novel adenoviral vector expressing GFP-TRAIL fusion protein from hTERT promoter

    PubMed Central

    2011-01-01

    Objective The objective of this study was to evaluate the anti-tumor effects of Ad/gTRAIL (an adenoviral vector in which expression of GFP and TRAIL is driven by a human telomerase reverse transcriptase promoter, hTERT) on malignant meningiomas and gliomas. Background Gliomas and meningiomas are the two most common types of human brain tumors. Currently there is no effective cure for recurrent malignant meningiomas or for gliomas. Ad/gTRAIL has been shown to be effective in killing selected lung, colon and breast cancer cells, but there have been no studies reporting its antitumor effects on malignant meningiomas. Therefore, we tested the antitumor effect of Ad/gTRAIL for the first time in human malignant meningioma and glioma cell lines, and in intracranial M6 and U87 xenografts. Methods Materials and Methods: Human malignant meningioma and glioma cells were infected with adenoviruses, Ad/gTRAIL and Ad/CMV-GFP. Cell viability was determined by proliferation assay. FACS analysis and quantification of TRAIL were used to measure apoptosis in these cells. We injected Ad/gTRAIL viruses in intracranial M6 and U87 xenografts, and measured the brain tumor volume, quantified apoptosis by TUNEL assay in the brain tumor tissue. Results Our studies demonstrate that in vitro/in vivo treatment with Ad/gTRAIL virus resulted in significant increase of TRAIL activity, and elicited a greater tumor cell apoptosis in malignant brain tumor cells as compared to treatment with the control, Ad/CMV-GFP virus without TRAIL activity. Conclusions We showed for the first time that adenovirus Ad/gTRAIL had significant antitumor effects against high grade malignant meningiomas as well as gliomas. Although more work needs to be done, our data suggests that Ad/gTRAIL has the potential to be useful as a tool against malignant brain tumors. PMID:22035360

  8. A tumor-targeting cRGD-EGFR siRNA conjugate and its anti-tumor effect on glioblastoma in vitro and in vivo.

    PubMed

    He, Shuai; Cen, Bohong; Liao, Lumin; Wang, Zhen; Qin, Yixin; Wu, Zhuomin; Liao, Wenjie; Zhang, Zhongyi; Ji, Aimin

    2017-11-01

    The epidermal growth factor receptor (EGFR) is an important anti-tumor target. The development of novel molecular-targeted anti-tumor drugs that can target the interior of tumor cells and specifically silence EGFR expression is valuable and promising. In this work, a promising anti-tumor conjugate comprising methoxy-modified EGFR siRNA and cyclic arginine-glycine-aspartic acid (cRGD) peptides, which selectively bind to αvβ3 integrins, was synthesized and examined. To prepare cRGD-EGFR siRNA (cRGD-siEGFR), cRGD was covalently conjugated to the 5'-end of an siRNA sense strand using a thiol-maleimide linker. The cellular uptake and cytotoxicity of cRGD-siEGFR in vitro were tested using an αvβ3-positive U87MG cell line. In vivo bio-distribution, anti-tumor activity, immunogenicity and toxicity were investigated in a nude mouse tumor model through repeated i.v. administration of cRGD-siEGFR (7 times over a 48 h interval). Analyses of in vitro data showed that cRGD-siEGFR silenced EGFR expression effectively, with high tumor targeting ability. Administration of cRGD-siEGFR to tumor-bearing nude mice led to significant inhibition of tumor growth, obvious reduction of EGFR expression and down-regulation of EGFR mRNA and protein in tumor tissue. Furthermore, serum biochemistry and pathological section evaluation did not indicate any serious toxicity of cRGD-siEGFR in vivo. cRGD-siEGFR is likely a promising candidate with high targeting ability, substantial anti-tumor effects and low toxicity in vitro and in vivo.

  9. A novel delivery vector for targeted delivery of the antiangiogenic drug paclitaxel to angiogenic blood vessels: TLTYTWS-conjugated PEG-PLA nanoparticles

    NASA Astrophysics Data System (ADS)

    Tan, Fei; Mo, Xiao-hui; Zhao, Jian; Liang, Hui; Chen, Zhong-jian; Wang, Xiu-li

    2017-02-01

    Antiangiogenesis has been widely accepted as an attractive strategy to combat tumor growth, invasion, and metastasis. An actively targeting nanoparticle-based drug delivery system (nano-DDS) would provide an alternative method to achieve antiangiogenic antitumor therapy. In the present study, our group fabricated novel nano-DDS, TLTYTWS (TS) peptide-modified poly(ethylene glycol)-poly(lactic acid) (PEG-PLA) nanoparticles (TS-NPs) encapsulating a drug with antiangiogenic potential, paclitaxel (Ptx) (TS-Ptx-NPs). The nanoparticles were uniformly spherical and had a unimodal particle size distribution and slightly negative zeta potential. TS-NPs accumulated significantly in human umbilical vein endothelial cells (HUVECs) via energy-dependent and caveolae- and lipid raft-mediated endocytosis and improved the antiproliferative, antimigratory, and antitube-forming abilities of paclitaxel in vitro. Following intravenous administration, TS-Ptx-NPs presented favorable pharmacokinetic profiles. Melanoma distribution assays confirmed that TS-NPs achieved higher accumulation and penetration at melanoma sites. These results collectively indicated that TLTYTWS-decorated nanoparticles can be considered to be a promising nano-DDS for chemotherapies targeting tumor angiogenesis and have great potential to improve the efficacy of antiangiogenic therapy in melanoma tumor-bearing nude mice.

  10. Spatially targeting Culex quinquefasciatus aquatic habitats on modified land cover for implementing an Integrated Vector Management (IVM) program in three villages within the Mwea Rice Scheme, Kenya

    PubMed Central

    Jacob, Benjamin G; Shililu, Josephat; Muturi, Ephantus J; Mwangangi, Joseph M; Muriu, Simon M; Funes, Jose; Githure, John; Regens, James L; Novak, Robert J

    2006-01-01

    limited control resources could be concentrated to reduce vector larval abundance. PMID:16684354

  11. Vaccine Design: Replication-Defective Adenovirus Vectors.

    PubMed

    Zhou, Xiangyang; Xiang, Zhiquan; Ertl, Hildegund C J

    2016-01-01

    Replication-defective adenovirus (Ad) vectors were initially developed for gene transfer for correction of genetic diseases. Although Ad vectors achieved high levels of transgene product expression in a variety of target cells, expression of therapeutic proteins was found to be transient as vigorous T cell responses directed to components of the vector as well as the transgene product rapidly eliminate Ad vector-transduced cells. This opened the use of Ad vectors as vaccine carriers and by now a multitude of preclinical as well as clinical studies has shown that Ad vectors induce very potent and sustained transgene product-specific T and B cell responses. This chapter provides guidance on developing E1-deleted Ad vectors based on available viral molecular clones. Specifically, it describes methods for cloning, viral rescue and purification as well as quality control studies.

  12. Fabrication and evaluation of tumor-targeted positive MRI contrast agent based on ultrasmall MnO nanoparticles.

    PubMed

    Huang, Haitao; Yue, Tao; Xu, Ke; Golzarian, Jafar; Yu, Jiahui; Huang, Jin

    2015-07-01

    Gd(III) chelate is currently used as positive magnetic resonance imaging (MRI) contrast agent in clinical diagnosis, but generally induces the risk of nephrogenic systemic fibrosis (NSF) due to the dissociated Gd(3+) from Gd(III) chelates. To develop a novel positive MRI contrast agent with low toxicity and high sensitivity, ultrasmall MnO nanoparticles were PEGylated via catechol-Mn chelation and conjugated with cRGD as active targeting function to tumor. Particularly, the MnO nanoparticles with a size of ca. 5nm were modified by α,β-poly(aspartic acid)-based graft polymer containing PEG and DOPA moieties and, meanwhile, conjugated with cRGD to produce the contrast agent with a size of ca. 100nm and a longitudinal relaxivity (r1) of 10.2mM(-1)S(-1). Such nanoscaled contrast agent integrated passive- and active-targeting function to tumor, and its efficient accumulation behavior in tumor was verified by in vivo distribution study. At the same time, the PEG moiety played a role of hydrophilic coating to improve the biocompatibility and stability under storing and physiological conditions, and especially might guarantee enough circulation time in blood. Moreover, in vivo MRI revealed a good and long-term effect of enhancing MRI signal for as-fabricated contrast agent while cell viability assay proved its acceptable cytotoxicity for MRI application. On the whole, the as-fabricated PEGylated and cRGD-functionalized contrast agent based on ultrasmall MnO nanoparticles showed a great potential to the T1-weighted MRI diagnosis of tumor. Crown Copyright © 2015. Published by Elsevier B.V. All rights reserved.

  13. ChAd63-MVA–vectored Blood-stage Malaria Vaccines Targeting MSP1 and AMA1: Assessment of Efficacy Against Mosquito Bite Challenge in Humans

    PubMed Central

    Sheehy, Susanne H; Duncan, Christopher JA; Elias, Sean C; Choudhary, Prateek; Biswas, Sumi; Halstead, Fenella D; Collins, Katharine A; Edwards, Nick J; Douglas, Alexander D; Anagnostou, Nicholas A; Ewer, Katie J; Havelock, Tom; Mahungu, Tabitha; Bliss, Carly M; Miura, Kazutoyo; Poulton, Ian D; Lillie, Patrick J; Antrobus, Richard D; Berrie, Eleanor; Moyle, Sarah; Gantlett, Katherine; Colloca, Stefano; Cortese, Riccardo; Long, Carole A; Sinden, Robert E; Gilbert, Sarah C; Lawrie, Alison M; Doherty, Tom; Faust, Saul N; Nicosia, Alfredo; Hill, Adrian VS; Draper, Simon J

    2012-01-01

    The induction of cellular immunity, in conjunction with antibodies, may be essential for vaccines to protect against blood-stage infection with the human malaria parasite Plasmodium falciparum. We have shown that prime-boost delivery of P. falciparum blood-stage antigens by chimpanzee adenovirus 63 (ChAd63) followed by the attenuated orthopoxvirus MVA is safe and immunogenic in healthy adults. Here, we report on vaccine efficacy against controlled human malaria infection delivered by mosquito bites. The blood-stage malaria vaccines were administered alone, or together (MSP1+AMA1), or with a pre-erythrocytic malaria vaccine candidate (MSP1+ME-TRAP). In this first human use of coadministered ChAd63-MVA regimes, we demonstrate immune interference whereby responses against merozoite surface protein 1 (MSP1) are dominant over apical membrane antigen 1 (AMA1) and ME-TRAP. We also show that induction of strong cellular immunity against MSP1 and AMA1 is safe, but does not impact on parasite growth rates in the blood. In a subset of vaccinated volunteers, a delay in time to diagnosis was observed and sterilizing protection was observed in one volunteer coimmunized with MSP1+AMA1—results consistent with vaccine-induced pre-erythrocytic, rather than blood-stage, immunity. These data call into question the utility of T cell-inducing blood-stage malaria vaccines and suggest that the focus should remain on high-titer antibody induction against susceptible antigen targets. PMID:23089736

  14. Dual TNF-α/IL-12p40 Interference as a Strategy to Protect Against Colitis Based on miR-16 Precursors With Macrophage Targeting Vectors.

    PubMed

    Huang, Zhen; Ma, Junting; Chen, Mengjie; Jiang, Haoyang; Fu, Yong; Gan, Jingjing; Dong, Lei; Zhang, Junfeng; Chen, Jiangning

    2015-10-01

    Cytokines are central components of the mucosal inflammatory responses that take place during the development of Crohn's disease. Cell-specific combination therapies against cytokines may lead to increased efficacy and even reduced side effects. Therefore, a colonic macrophage-specific therapy using miR-16 precursors that can target both TNF-α and IL-12p40 was tested for its efficacy in experimental colitic mice. Galactosylated low molecular weight chitosan (G-LMWC) associated with miR-16 precursors were intracolonically injected into mice. The cellular localization of miR-16 precursors was determined. The therapeutic effects and possible mechanism were further studied in 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitic mice. The results show that specific upregulation of miR-16 level in colonic macrophages significantly reduces TNF-α and IL-12p40 expression, which could suppress the associated mucosal inflammation and ultimately result in the relief of colitic symptoms. This strategy, based on the dual silencing of colonic macrophage-specific cytokines, represents a potential therapeutic approach that may be valuable for colitis therapy.

  15. A report on the indoor residual spraying (IRS) in the control of Phlebotomus argentipes, the vector of visceral leishmaniasis in Bihar (India): an initiative towards total elimination targeting 2015 (Series-1).

    PubMed

    Kumar, V; Kesari, S; Dinesh, D S; Tiwari, A K; Kumar, A J; Kumar, R; Singh, V P; Das, P

    2009-09-01

    Visceral leishmaniasis, commonly known as kala-azar is endemic in Bihar state, India. Current vector control programme in Bihar focuses mainly on spraying the sandfly infested dwellings with DDT. The Government of India in collaboration with WHO has fixed the target 2015 for total elimination of kala-azar. The present study was carried out to see the impact of DDT and improved IEC in the containment of vector density vis-à-vis disease transmission. Before the start of the spraying operations training was imparted to all the medical and paramedical personnel regarding the methods of spraying operations. Pre- and post-sandfly density was monitored in four selected districts. Incidences of kala-azar cases were compared for pre- and post-spray periods. Social acceptability and perceptions of households was collected through questionnaires from 500 randomly selected households in the study districts. House index in three study districts reduced considerably during post-spray when compared to pre-spray. Kala-azar incidence in many districts was reduced after the DDT spray. Either partial or complete refusal was reported in 14.4%, while 35% were not satisfied with the suspension concentration and coverage; and 46.6% were found satisfied with the spraying procedure. Strengthening the IEC activities to sensitise the community, proper training of health personnel, monitoring of spray, good surveillance, proper treatment of cases and two rounds of DDT spray with good coverage in the endemic districts up to three years are essential to achieve the desired total elimination of kala-azar in Bihar state.

  16. Episomal vectors for gene therapy.

    PubMed

    Ehrhardt, Anja; Haase, Rudolf; Schepers, Aloys; Deutsch, Manuel J; Lipps, Hans Joachim; Baiker, Armin

    2008-06-01

    The increasing knowledge of the molecular and genetic background of many different human diseases has led to the vision that genetic engineering might be used one day for their phenotypic correction. The main goal of gene therapy is to treat loss-of-function genetic disorders by delivering correcting therapeutic DNA sequences into the nucleus of a cell, allowing its long-term expression at physiologically relevant levels. Manifold different vector systems for the therapeutic gene delivery have been described over the recent years. They all have their individual advantages but also their individual limitations and must be judged on a careful risk/benefit analysis. Integrating vector systems can deliver genetic material to a target cell with high efficiency enabling long-term expression of an encoded transgene. The main disadvantage of integrating vector systems, however, is their potential risk of causing insertional mutagenesis. Episomal vector systems have the potential to avoid these undesired side effects, since they behave as separate extrachromosomal elements in the nucleus of a target cell. Within this article we present a comprehensive survey of currently available episomal vector systems for the genetic modification of mammalian cells. We will discuss their advantages and disadvantages and their applications in the context of basic research, biotechnology and gene therapy.

  17. Green-synthesized CdS nano-pesticides: Toxicity on young instars of malaria vectors and impact on enzymatic activities of the non-target mud crab Scylla serrata.

    PubMed

    Sujitha, Vasu; Murugan, Kadarkarai; Dinesh, Devakumar; Pandiyan, Amuthvalli; Aruliah, Rajasekar; Hwang, Jiang-Shiou; Kalimuthu, Kandasamy; Panneerselvam, Chellasamy; Higuchi, Akon; Aziz, Al Thabiani; Kumar, Suresh; Alarfaj, Abdullah A; Vaseeharan, Baskaralingam; Canale, Angelo; Benelli, Giovanni

    2017-07-01

    Currently, nano-formulated mosquito larvicides have been widely proposed to control young instars of malaria vector populations. However, the fate of nanoparticles in the aquatic environment is scarcely known, with special reference to the impact of nanoparticles on enzymatic activity of non-target aquatic invertebrates. In this study, we synthesized CdS nanoparticles using a green protocol relying on the cheap extract of Valoniopsis pachynema algae. CdS nanoparticles showed high toxicity on young instars of the malaria vectors Anopheles stephensi and A. sundaicus. The antimalarial activity of the nano-synthesized product against chloroquine-resistant (CQ-r) Plasmodium falciparum parasites was investigated. From a non-target perspective, we focused on the impact of this novel nano-pesticide on antioxidant enzymes acetylcholinesterase (AChE) and glutathione S-transferase (GST) activities of the mud crab Scylla serrata. The characterization of nanomaterials was carried out by UV-vis and FTIR spectroscopy, as well as SEM and XRD analyses. In mosquitocidal assays, LC50 of V. pachynema-synthesized CdS nanoparticles on A. stephensi ranged from 16.856 (larva I), to 30.301μg/ml (pupa), while for An. sundaicus they ranged from 13.584 to 22.496μg/ml. The antiplasmodial activity of V. pachynema extract and CdS nanoparticles was evaluated against CQ-r and CQ-sensitive (CQ-s) strains of Plasmodium falciparum. IC50 of V. pachynema extract was 58.1μg/ml (CQ-s) and 71.46μg/ml (CQ-r), while nano-CdS IC50 was 76.14μg/ml (CQ-s) and 89.21μg/ml (CQ-r). In enzymatic assays, S. serrata crabs were exposed to sub-lethal concentrations, i.e. 4, 6 and 8μg/ml of CdS nanoparticles, assessing changes in GST and AChE activity after 16days. We observed significantly higher activity of GST, if compared to the control, during the whole experiment period. In addition, a single treatment with CdS nanoparticles led to a significant decrease in AChE activity over time. The toxicity of Cd

  18. [64Cu-NOTA-8-Aoc-BBN(7-14)NH2] targeting vector for positron-emission tomography imaging of gastrin-releasing peptide receptor-expressing tissues.

    PubMed

    Prasanphanich, Adam F; Nanda, Prasant K; Rold, Tammy L; Ma, Lixin; Lewis, Michael R; Garrison, Jered C; Hoffman, Timothy J; Sieckman, Gary L; Figueroa, Said D; Smith, Charles J

    2007-07-24

    Radiolabeled peptides hold promise as diagnostic/therapeutic targeting vectors for specific human cancers. We report the design and development of a targeting vector, [(64)Cu-NOTA-8-Aoc-BBN(7-14)NH(2)] (NOTA = 1,4,7-triazacyclononane-1,4,7-triacetic acid, 8-Aoc = 8-aminooctanoic acid, and BBN = bombesin), having very high selectivity and affinity for the gastrin-releasing peptide receptor (GRPr). GRPrs are expressed on a variety of human cancers, including breast, lung, pancreatic, and prostate, making this a viable approach toward site-directed localization or therapy of these human diseases. In this study, [NOTA-X-BBN(7-14)NH(2)] conjugates were synthesized, where X = a specific pharmacokinetic modifier. The IC(50) of [NOTA-8-Aoc-BBN(7-14)NH(2)] was determined by a competitive displacement cell-binding assay in PC-3 human prostate cancer cells using (125)I-[Tyr(4)]-BBN as the displacement ligand. An IC(50) of 3.1 +/- 0.5 nM was obtained, demonstrating high binding affinity of [NOTA-8-Aoc-BBN] for the GRPr. [(64)Cu-NOTA-X-BBN] conjugates were prepared by the reaction of (64)CuCl(2) with peptides in buffered aqueous solution. In vivo studies of [(64)Cu-NOTA-8-Aoc-BBN(7-14)NH(2)] in tumor-bearing PC-3 mouse models indicated very high affinity of conjugate for the GRPr. Uptake of conjugate in tumor was 3.58 +/- 0.70% injected dose (ID) per g at 1 h postintravenous injection (p.i.). Minimal accumulation of radioactivity in liver tissue (1.58 +/- 0.40% ID per g, 1 h p.i.) is indicative of rapid renal-urinary excretion and suggests very high in vivo kinetic stability of [(64)Cu-NOTA-8-Aoc-BBN(7-14)NH(2)] with little or no in vivo dissociation of (64)Cu(2+) from the NOTA chelator. Kidney accumulation at 1 h p.i. was 3.79 +/- 1.09% ID per g. Molecular imaging studies in GRPr-expressing tumor models produced high-contrast, high-quality micro-positron-emission tomography images.

  19. Rotations with Rodrigues' Vector

    ERIC Educational Resources Information Center

    Pina, E.

    2011-01-01

    The rotational dynamics was studied from the point of view of Rodrigues' vector. This vector is defined here by its connection with other forms of parametrization of the rotation matrix. The rotation matrix was expressed in terms of this vector. The angular velocity was computed using the components of Rodrigues' vector as coordinates. It appears…

  20. Rotations with Rodrigues' Vector

    ERIC Educational Resources Information Center

    Pina, E.

    2011-01-01

    The rotational dynamics was studied from the point of view of Rodrigues' vector. This vector is defined here by its connection with other forms of parametrization of the rotation matrix. The rotation matrix was expressed in terms of this vector. The angular velocity was computed using the components of Rodrigues' vector as coordinates. It appears…

  1. First-in-Human Treatment With a Dendritic Cell-targeting Lentiviral Vector-expressing NY-ESO-1, LV305, Induces Deep, Durable Response in Refractory Metastatic Synovial Sarcoma Patient.

    PubMed

    Pollack, Seth M; Lu, Hailing; Gnjatic, Sacha; Somaiah, Neeta; O'Malley, Ryan B; Jones, Robin L; Hsu, Frank J; Ter Meulen, Jan

    2017-10-01

    Effective induction of antitumor T cells is a pivotal goal of cancer immunotherapy. To this end, lentiviral vectors (LV) are uniquely poised to directly prime CD8 T-cell responses via transduction of dendritic cells in vivo and have shown promise as active cancer therapeutics in preclinical tumor models. However, until now, significant barriers related to production and regulation have prevented their widespread use in the clinic. We developed LV305, a dendritic cell-targeting, integration-deficient, replication incompetent LV from the ZVex platform, encoding the full-length cancer-testis antigen NY-ESO-1. LV305 is currently being evaluated in phase 1 and 2 trials in metastatic recurrent cancer patients with NY-ESO-1 positive solid tumors as a single agent and in combination with anti-PD-L1. Here we report on the first patient treated with LV305, a young woman with metastatic, recurrent, therapy-refractive NY-ESO-1 synovial sarcoma. The patient developed a robust NY-ESO-1-specific CD4 and CD8 T-cell response after 3 intradermal injections with LV305, and subsequently over 85% disease regression that is continuing for >2.5 years posttherapy. No adverse events >grade 2 occurred. This case demonstrates that LV305 can be safely administered and has the potential to induce a significant clinical benefit and immunologic response in a patient with advanced stage cancer.

  2. Viral vectors for vascular gene therapy

    PubMed Central

    Fischer, Lukas; Preis, Meir; Weisz, Anat; Koren, Belly; Lewis, Basil S; Flugelman, Moshe Y

    2002-01-01

    Vascular gene therapy is the focus of multiple experimental and clinical research efforts. While several genes with therapeutic potential have been identified, the best method of gene delivery is unknown. Viral vectors have the capacity to transfer genes at high efficiency rates. Several viral-based vectors have been used in experimental vascular gene therapy for in vivo and ex vivo gene transfer. Adenoviral-based vectors are being used for the induction of angiogenesis in phase 1 and 2 clinical trials. In the present review, the characteristics of the ‘ideal’ viral vector are discussed and the major types of viral vectors used in vascular gene transfer are reviewed. Basic knowledge of the use of viral vectors for direct in vivo gene transfer (adenoviral-based vectors, etc) and for ex vivo gene transfer (retroviral-based vectors) is provided. New developments in the field of viral vectorology, such as pseudotyping of retroviral vectors and targeting of other viral vectors to a specific cell type, will enhance the more rapid transition of vascular gene therapy from the experimental arena to the clinical setting. PMID:19649233

  3. Light axial vector mesons

    NASA Astrophysics Data System (ADS)

    Chen, Kan; Pang, Cheng-Qun; Liu, Xiang; Matsuki, Takayuki

    2015-04-01

    Inspired by the abundant experimental observation of axial-vector states, we study whether the observed axial-vector states can be categorized into the conventional axial-vector meson family. In this paper we carry out an analysis based on the mass spectra and two-body Okubo-Zweig-Iizuka-allowed decays. Besides testing the possible axial-vector meson assignments, we also predict abundant information for their decays and the properties of some missing axial-vector mesons, which are valuable for further experimental exploration of the observed and predicted axial-vector mesons.

  4. Reduced Vector Preisach Model

    NASA Technical Reports Server (NTRS)

    Patel, Umesh D.; Torre, Edward Della; Day, John H. (Technical Monitor)

    2002-01-01

    A new vector Preisach model, called the Reduced Vector Preisach model (RVPM), was developed for fast computations. This model, derived from the Simplified Vector Preisach model (SVPM), has individual components that like the SVPM are calculated independently using coupled selection rules for the state vector computation. However, the RVPM does not require the rotational correction. Therefore, it provides a practical alternative for computing the magnetic susceptibility using a differential approach. A vector version, using the framework of the DOK model, is implemented. Simulation results for the reduced vector Preisach model are also presented.

  5. Building mosaics of therapeutic plasmid gene vectors.

    PubMed

    Tolmachov, Oleg E

    2011-12-01

    Plasmids are circular or linear DNA molecules propagated extra-chromosomally in bacteria. Evolution shaped plasmids are inherently mosaic structures with individual functional units represented by distinct segments in the plasmid genome. The patchwork of plasmid genetic modules is a convenient template and a model for the generation of artificial plasmids used as vehicles for gene delivery into human cells. Plasmid gene vectors are an important tool in gene therapy and in basic biomedical research, where these vectors offer efficient transgene expression in many settings in vitro and in vivo. Plasmid vectors can be attached to nuclear directing ligands or transferred by electroporation as naked DNA to deliver the payload genes to the nuclei of the target cells. Transgene expression silencing by plasmid sequences of bacterial origin and immune stimulation by bacterial unmethylated CpG motifs can be avoided by the generation of plasmid-based minimized DNA vectors, such as minicircles. Systems of efficient site-specific integration into human chromosomes and stable episomal maintenance in human cells are being developed for further reduction of the chances for transgene silencing. The successful generation of plasmid vectors is governed by a number of vector design rules, some of which are common to all gene vectors, while others are specific to plasmid vectors. This review is focused both on the guiding principles and on the technical know-how of plasmid gene vector design.

  6. Surface-engineering of lentiviral vectors.

    PubMed

    Verhoeyen, Els; Cosset, François-Loïc

    2004-02-01

    Vectors derived from retroviridae offer particularly flexible properties in gene transfer applications given the numerous possible associations of various viral surface glycoproteins (determining cell tropism) with different types of retroviral cores (determining genome replication and integration). Lentiviral vectors should be preferred gene delivery vehicles over vectors derived from onco-retroviruses such as murine leukemia viruses (MLVs) that cannot transduce non-proliferating target cells. Generating lentiviral vectors pseudotyped with different viral glycoproteins (GPs) may modulate the physicochemical properties of the vectors, their interaction with the host immune system and their host range. There are however important gene transfer restrictions to some non-proliferative tissues or cell types and recent studies have shown that progenitor hematopoietic stem cells in G(0), non-activated primary blood lymphocytes or monocytes were not transducible by lentiviral vectors. Moreover, lentiviral vectors that have the capacity to deliver transgenes into specific tissues are expected to be of great value for various gene transfer applications in vivo. Several innovative approaches have been explored to overcome such problems that have given rise to novel concepts in the field and have provided promising results in preliminary evaluations in vivo. Here we review the different approaches explored to upgrade lentiviral vectors, aiming at developing vectors suitable for in vivo gene delivery.

  7. First-in-Human Treatment With a Dendritic Cell-targeting Lentiviral Vector-expressing NY-ESO-1, LV305, Induces Deep, Durable Response in Refractory Metastatic Synovial Sarcoma Patient.

    PubMed

    Pollack, Seth M; Lu, Hailing; Gnjatic, Sacha; Somaiah, Neeta; O'Malley, Ryan B; Jones, Robin L; Hsu, Frank J; Ter Meulen, Jan

    2017-09-18

    Effective induction of antitumor T cells is a pivotal goal of cancer immunotherapy. To this end, lentiviral vectors (LV) are uniquely poised to directly prime CD8 T-cell responses via transduction of dendritic cells in vivo and have shown promise as active cancer therapeutics in preclinical tumor models. However, until now, significant barriers related to production and regulation have prevented their widespread use in the clinic. We developed LV305, a dendritic cell-targeting, integration-deficient, replication incompetent LV from the ZVex platform, encoding the full-length cancer-testis antigen NY-ESO-1. LV305 is currently being evaluated in phase 1 and 2 trials in metastatic recurrent cancer patients with NY-ESO-1 positive solid tumors as a single agent and in combination with anti-PD-L1. Here we report on the first patient treated with LV305, a young woman with metastatic, recurrent, therapy-refractive NY-ESO-1 synovial sarcoma. The patient developed a robust NY-ESO-1-specific CD4 and CD8 T-cell response after 3 intradermal injections with LV305, and subsequently over 85% disease regression that is continuing for >2.5 years posttherapy. No adverse events >grade 2 occurred. This case demonstrates that LV305 can be safely administered and has the potential to induce a significant clinical benefit and immunologic response in a patient with advanced stage cancer.This is an open-access article distributed under the terms of the Creative Commons Attribution-Non Commercial-No Derivatives License 4.0 (CCBY-NC-ND), where it is permissible to download and share the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal. http://creativecommons.org/licenses/by-nc-nd/4.0/.

  8. Understanding Singular Vectors

    ERIC Educational Resources Information Center

    James, David; Botteron, Cynthia

    2013-01-01

    matrix yields a surprisingly simple, heuristical approximation to its singular vectors. There are correspondingly good approximations to the singular values. Such rules of thumb provide an intuitive interpretation of the singular vectors that helps explain why the SVD is so…

  9. Understanding Singular Vectors

    ERIC Educational Resources Information Center

    James, David; Botteron, Cynthia

    2013-01-01

    matrix yields a surprisingly simple, heuristical approximation to its singular vectors. There are correspondingly good approximations to the singular values. Such rules of thumb provide an intuitive interpretation of the singular vectors that helps explain why the SVD is so…

  10. Insulated Foamy Viral Vectors

    PubMed Central

    Browning, Diana L.; Collins, Casey P.; Hocum, Jonah D.; Leap, David J.; Rae, Dustin T.; Trobridge, Grant D.

    2016-01-01

    Retroviral vector-mediated gene therapy is promising, but genotoxicity has limited its use in the clinic. Genotoxicity is highly dependent on the retroviral vector used, and foamy viral (FV) vectors appear relatively safe. However, internal promoters may still potentially activate nearby genes. We developed insulated FV vectors, using four previously described insulators: a version of the well-studied chicken hypersensitivity site 4 insulator (650cHS4), two synthetic CCCTC-binding factor (CTCF)-based insulators, and an insulator based on the CCAAT box-binding transcription factor/nuclear factor I (7xCTF/NF1). We directly compared these insulators for enhancer-blocking activity, effect on FV vector titer, and fidelity of transfer to both proviral long terminal repeats. The synthetic CTCF-based insulators had the strongest insulating activity, but reduced titers significantly. The 7xCTF/NF1 insulator did not reduce titers but had weak insulating activity. The 650cHS4-insulated FV vector was identified as the overall most promising vector. Uninsulated and 650cHS4-insulated FV vectors were both significantly less genotoxic than gammaretroviral vectors. Integration sites were evaluated in cord blood CD34+ cells and the 650cHS4-insulated FV vector had fewer hotspots compared with an uninsulated FV vector. These data suggest that insulated FV vectors are promising for hematopoietic stem cell gene therapy. PMID:26715244

  11. Restart 68000 vector remapping

    SciTech Connect

    Gustin, J.

    1984-05-03

    The circuit described allows power-on-reset (POR) vector fetch from ROM for a 68000 microprocessor. It offers programmability of exception vectors, including the restart vector. This method eliminates the need for high-resolution, address-decoder peripheral circuitry.

  12. Rhotrix Vector Spaces

    ERIC Educational Resources Information Center

    Aminu, Abdulhadi

    2010-01-01

    By rhotrix we understand an object that lies in some way between (n x n)-dimensional matrices and (2n - 1) x (2n - 1)-dimensional matrices. Representation of vectors in rhotrices is different from the representation of vectors in matrices. A number of vector spaces in matrices and their properties are known. On the other hand, little seems to be…

  13. Rhotrix Vector Spaces

    ERIC Educational Resources Information Center

    Aminu, Abdulhadi

    2010-01-01

    By rhotrix we understand an object that lies in some way between (n x n)-dimensional matrices and (2n - 1) x (2n - 1)-dimensional matrices. Representation of vectors in rhotrices is different from the representation of vectors in matrices. A number of vector spaces in matrices and their properties are known. On the other hand, little seems to be…

  14. Insulated Foamy Viral Vectors.

    PubMed

    Browning, Diana L; Collins, Casey P; Hocum, Jonah D; Leap, David J; Rae, Dustin T; Trobridge, Grant D

    2016-03-01

    Retroviral vector-mediated gene therapy is promising, but genotoxicity has limited its use in the clinic. Genotoxicity is highly dependent on the retroviral vector used, and foamy viral (FV) vectors appear relatively safe. However, internal promoters may still potentially activate nearby genes. We developed insulated FV vectors, using four previously described insulators: a version of the well-studied chicken hypersensitivity site 4 insulator (650cHS4), two synthetic CCCTC-binding factor (CTCF)-based insulators, and an insulator based on the CCAAT box-binding transcription factor/nuclear factor I (7xCTF/NF1). We directly compared these insulators for enhancer-blocking activity, effect on FV vector titer, and fidelity of transfer to both proviral long terminal repeats. The synthetic CTCF-based insulators had the strongest insulating activity, but reduced titers significantly. The 7xCTF/NF1 insulator did not reduce titers but had weak insulating activity. The 650cHS4-insulated FV vector was identified as the overall most promising vector. Uninsulated and 650cHS4-insulated FV vectors were both significantly less genotoxic than gammaretroviral vectors. Integration sites were evaluated in cord blood CD34(+) cells and the 650cHS4-insulated FV vector had fewer hotspots compared with an uninsulated FV vector. These data suggest that insulated FV vectors are promising for hematopoietic stem cell gene therapy.

  15. Construction of hydroxypropyl-β-cyclodextrin copolymer nanoparticles and targeting delivery of paclitaxel

    NASA Astrophysics Data System (ADS)

    Miao, Qinghua; Li, Suping; Han, Siyuan; Wang, Zhi; Wu, Yan; Nie, Guangjun

    2012-08-01

    A novel amphiphilic copolymer with p-maleimidophenyl isocyanate-hydroxypropyl-β-cyclodextrin-polylactide-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine to generate copolymer nanoparticles (NPs) has been designed. In order to develop an active targeting system, integrin αvβ3-specific targeting peptide cyclo(Arg-Gly-Asp-D-Phe-Cys), cRGD, was conjugated to the surface of NPs (NPs-RGD). These NPs were used to encapsulate anti-tumor drug, paclitaxel. The resulting NPs exhibited high drug-loading capacity and controlled drug release in vitro at acidic pH. In vitro cytotoxicity assay demonstrates that paclitaxel-loaded NPs-RGD significantly inhibited B16 tumor cell (high αvβ3) proliferation relative to free paclitaxel and paclitaxel-loaded NPs at high concentrations. Paclitaxel-loaded NPs-RGD localized mainly in lysosomes in B16 cells as revealed by confocal microscopy. These results suggest a novel strategy for fabrication—functionalizing hydroxypropyl-β-cyclodextrin copolymer nanoparticles for targeting delivery of paclitaxel to integrin αvβ3-rich tumor cells. These nanocarriers can be readily extended to couple other bioactive molecules for active targeting and delivery of various chemotherapeutic drugs.

  16. Strategies for retargeted gene delivery using vectors derived from lentiviruses.

    PubMed

    Bartosch, Birke; Cosset, Francois-Loic

    2004-12-01

    With the development of the first viral vector systems 20 years ago [Mann et al., 1983; Watanabe and Temin, 1983] gene therapy strategies have come to the forefront of novel therapeutics [Cavazzana-Calvo et al., 2000]. A deeper understanding of vector biology and the molecular mechanisms of disease alongside tremendous advances in vector technology have significantly advanced the field of human gene therapy. Over the last few years several challenges needed to be overcome in order to bring gene therapy strategies closer to the clinic. These hurdles include the preparation of large amounts of stable, high titre vectors, minimising vector-related immunology and last but not least targeting infection and transgene expression to tissue or cells, which in many cases are not or only slowly dividing. Viral vectors are useful vehicles for the delivery of foreign genes into target cells, and retroviral vectors have been popular because of their ability to integrate into the host cell genome and maintain persistent gene expression. Moreover, lentiviruses, members of the retroviral family, have the ability to infect cells at both mitotic and post-mitotic stages of the cell cycle thus opening up the possibility to target non-dividing target cells and tissues. Human immunodeficiency virus (HIV) based vectors have been used in vitro and in vivo in a number of situations, however, safety concerns still exist, and therefore the development of vector systems based on primate as well as non-primate lentiviruses is ongoing. Concomitantly with lentiviral vector design, much has been learned about the incorporation of heterologous env proteins on lentiviral cores in order to combine specific targeting properties of envelope glycoproteins with the biological properties of lentiviral vectors. In this review article we will give an overview over advantages lentiviral vector systems offer. We will then discuss the current state of our understanding of the structure and function of viral

  17. Integrated strategy for the production of therapeutic retroviral vectors.

    PubMed

    Carrondo, Manuel; Panet, Amos; Wirth, Dagmar; Coroadinha, Ana Sofia; Cruz, Pedro; Falk, Haya; Schucht, Roland; Dupont, Francis; Geny-Fiamma, Cécile; Merten, Otto-Wilhelm; Hauser, Hansjörg

    2011-03-01

    The broad application of retroviral vectors for gene delivery is still hampered by the difficulty to reproducibly establish high vector producer cell lines generating sufficient amounts of highly concentrated virus vector preparations of high quality. To enhance the process for producing clinically relevant retroviral vector preparations for therapeutic applications, we have integrated novel and state-of-the-art technologies in a process that allows rapid access to high-efficiency vector-producing cells and consistent production, purification, and storage of retroviral vectors. The process has been designed for various types of retroviral vectors for clinical application and to support a high-throughput process. New modular helper cell lines that permit rapid insertion of DNA encoding the therapeutic vector of interest at predetermined, optimal chromosomal loci were developed to facilitate stable and high vector production levels. Packaging cell lines, cultivation methods, and improved medium composition were coupled with vector purification and storage process strategies that yield maximal vector infectivity and stability. To facilitate GMP-grade vector production, standard of operation protocols were established. These processes were validated by production of retroviral vector lots that drive the expression of type VII collagen (Col7) for the treatment of a skin genetic disease, dystrophic epidermolysis bullosa. The potential efficacy of the Col7-expressing vectors was finally proven with newly developed systems, in particular in target primary keratinocyte cultures and three-dimensional skin tissues in organ culture.

  18. Hard Exclusive Vector Meson Leptoproduction At HERMES

    SciTech Connect

    Golembiovskaya, M.

    2011-07-15

    The HERMES experiment at DESY, Hamburg collected a set of data on hard exclusive vector meson ({rho}{sup 0}{phi},{omega}) leptoproduction using the 27.6 GeV longitudinally polarized lepton beam of HERA accelerator and longitudinally or transversely polarized or unpolarized gas targets. Measurements of exclusive vector meson production provide access to the structure of the nucleon since the process can be described in terms of Generalized Parton Distributions (GPDs). An overview of the HERMES results on exclusive vector mesons production is presented.

  19. Mosquito vectors of dog heartworm in the United States: vector status and factors influencing transmission efficiency.

    PubMed

    Ledesma, Nicholas; Harrington, Laura

    2011-11-01

    Dog heartworm (Dirofilaria immitis) is dependent on mosquito vectors for its maintenance and transmission among vertebrate hosts. Consequently, D. immitis abundance and distribution are closely linked with mosquito vector biology and ecology. Information on the important dog heartworm vectors in the United States is limited and no comprehensive surveillance of dog heartworm in US mosquitoes has been undertaken to date. Here, we review information gleaned from a number of field surveys documenting heartworm presence in wild mosquito populations as well as laboratory assessments of mosquito vector capacity. Various biological and ecological factors likely contribute to the relative importance of different vector species. We describe some of these factors, rank the leading criteria for efficient vectors, and present the most likely vector species found across the United States. Considering the recent emergence of drug resistance among D. immitis strains, practical knowledge of heartworm vector biology and control should be incorporated into heartworm disease management programs. We conclude by proposing that heartworm control would benefit by targeting mosquito vectors, and we suggest ways in which veterinarians can incorporate the recognition of vector importance into heartworm prevention recommendations imparted to clients. Copyright © 2011 Elsevier Inc. All rights reserved.

  20. Gene therapy using retrovirus vectors: vector development and biosafety at clinical trials.

    PubMed

    Doi, Knayo; Takeuchi, Yasuhiro

    2015-01-01

    Retrovirus vectors (gammaretroviral and lentiviral vectors) have been considered as promising tools to transfer therapeutic genes into patient cells because they can permanently integrate into host cellular genome. To treat monogenic, inherited diseases, retroviral vectors have been used to add correct genes into patient cells. Conventional gammaretroviral vectors achieved successful results in clinical trials: treated patients had therapeutic gene expression in target cells and had improved symptoms of diseases. However, serious side-effects of leukemia occurred, caused by retroviral insertional mutagenesis (IM). These incidences stressed the importance of monitoring vector integration sites in patient cells as well as of re-consideration on safer vectors. More recently lentiviral vectors which can deliver genes into non-dividing cells started to be used in clinical trials including neurological disorders, showing their efficacy. Vector integration site analysis revealed that lentiviruses integrate less likely to near promoter regions of oncogenes than gammaretroviruses and no adverse events have been reported in lentiviral vector-mediated gene therapy clinical trials. Therefore lentiviral vectors have promises to be applied to a wide range of common diseases in near future. For example, T cells from cancer patients were transduced to express chimeric T cell receptors recognizing their tumour cells enhancing patients' anti-cancer immunity.

  1. Biosafety Features of Lentiviral Vectors

    PubMed Central

    Schambach, Axel; Zychlinski, Daniela; Ehrnstroem, Birgitta

    2013-01-01

    Abstract Over the past decades, lentiviral vectors have evolved as a benchmark tool for stable gene transfer into cells with a high replicative potential. Their relatively flexible genome and ability to transduce many forms of nondividing cells, combined with the potential for cell-specific pseudotyping, provides a rich resource for numerous applications in experimental platforms and therapeutic settings. Here, we give an overview of important biosafety features of lentiviral vectors, with detailed discussion of (i) the principles of the lentiviral split-genome design used for the construction of packaging cells; (ii) the relevance of modifications introduced into the lentiviral long terminal repeat (deletion of enhancer/promoter sequences and introduction of insulators); (iii) the basic features of mRNA processing, including the Rev/Rev-responsive element (RRE) interaction and the modifications of the 3′ untranslated region of lentiviral vectors with various post-transcriptional regulatory elements affecting transcriptional termination, polyadenylation, and differentiation-specific degradation of mRNA; and (iv) the characteristic integration pattern with the associated risk of transcriptional interference with cellular genes. We conclude with considerations regarding the importance of cell targeting via envelope modifications. Along this course, we address canonical biosafety issues encountered with any type of viral vector: the risks of shedding, mobilization, germline transmission, immunogenicity, and insertional mutagenesis. PMID:23311447

  2. Biosafety features of lentiviral vectors.

    PubMed

    Schambach, Axel; Zychlinski, Daniela; Ehrnstroem, Birgitta; Baum, Christopher

    2013-02-01

    Over the past decades, lentiviral vectors have evolved as a benchmark tool for stable gene transfer into cells with a high replicative potential. Their relatively flexible genome and ability to transduce many forms of nondividing cells, combined with the potential for cell-specific pseudotyping, provides a rich resource for numerous applications in experimental platforms and therapeutic settings. Here, we give an overview of important biosafety features of lentiviral vectors, with detailed discussion of (i) the principles of the lentiviral split-genome design used for the construction of packaging cells; (ii) the relevance of modifications introduced into the lentiviral long terminal repeat (deletion of enhancer/promoter sequences and introduction of insulators); (iii) the basic features of mRNA processing, including the Rev/Rev-responsive element (RRE) interaction and the modifications of the 3' untranslated region of lentiviral vectors with various post-transcriptional regulatory elements affecting transcriptional termination, polyadenylation, and differentiation-specific degradation of mRNA; and (iv) the characteristic integration pattern with the associated risk of transcriptional interference with cellular genes. We conclude with considerations regarding the importance of cell targeting via envelope modifications. Along this course, we address canonical biosafety issues encountered with any type of viral vector: the risks of shedding, mobilization, germline transmission, immunogenicity, and insertional mutagenesis.

  3. A targeted nanoglobular contrast agent from host-guest self-assembly for MR cancer molecular imaging

    PubMed Central

    Zhou, Zhuxian; Han, Zhen; Lu, Zheng-Rong

    2016-01-01

    The clinical application of nanoparticular Gd(III) based contrast agents for tumor molecular MRI has been hindered by safety concerns associated with prolonged tissue retention, although they can produce strong tumor enhancement. In this study, a targeted well-defined cyclodextrin-based nanoglobular contrast agent was developed through self-assembly driven by host-guest interactions for safe and effective cancer molecular MRI. Multiple β-cyclodextrins attached POSS (polyhedral oligomeric silsesquioxane) nanoglobule was used as host molecule. Adamantane–modified macrocyclic Gd(III) contrast agent, cRGD (cyclic RGDfK peptide) targeting ligand and fluorescent probe was used as guest molecules. The targeted host-guest nanoglobular contrast agent cRGD-POSS-βCD-(DOTA-Gd) specifically bond to αvβ3 integrin in malignant 4T1 breast tumor and provided greater contrast enhancement than the corresponding non-targeted agent. The agent also provided significant fluorescence signal in tumor tissue. The histological analysis of the tumor tissue confirmed its specific and effective targeting to αvβ3 integrin. The targeted imaging agent has a potential for specific cancer molecular MR and fluorescent imaging. PMID:26874280

  4. A targeted nanoglobular contrast agent from host-guest self-assembly for MR cancer molecular imaging.

    PubMed

    Zhou, Zhuxian; Han, Zhen; Lu, Zheng-Rong

    2016-04-01

    The clinical application of nanoparticular Gd(III) based contrast agents for tumor molecular MRI has been hindered by safety concerns associated with prolonged tissue retention, although they can produce strong tumor enhancement. In this study, a targeted well-defined cyclodextrin-based nanoglobular contrast agent was developed through self-assembly driven by host-guest interactions for safe and effective cancer molecular MRI. Multiple β-cyclodextrins attached POSS (polyhedral oligomeric silsesquioxane) nanoglobule was used as host molecule. Adamantane-modified macrocyclic Gd(III) contrast agent, cRGD (cyclic RGDfK peptide) targeting ligand and fluorescent probe was used as guest molecules. The targeted host-guest nanoglobular contrast agent cRGD-POSS-βCD-(DOTA-Gd) specifically bond to αvβ3 integrin in malignant 4T1 breast tumor and provided greater contrast enhancement than the corresponding non-targeted agent. The agent also provided significant fluorescence signal in tumor tissue. The histological analysis of the tumor tissue confirmed its specific and effective targeting to αvβ3 integrin. The targeted imaging agent has a potential for specific cancer molecular MR and fluorescent imaging. Copyright © 2016 Elsevier Ltd. All rights reserved.

  5. Beyond Gene Delivery: Strategies to Engineer the Surfaces of Viral Vectors.

    PubMed

    Capasso, Cristian; Hirvinen, Mari; Cerullo, Vincenzo

    2013-12-04

    Viral vectors have been extensively studied due to their great transduction efficiency compared to non-viral vectors. These vectors have been used extensively in gene therapy, enabling the comprehension of, not only the advantages of these vectors, but also the limitations, such as the activation of the immune system after vector administration. Moreover, the need to control the target of the vector has led to the development of chemical and non-chemical modifications of the vector surface, allowing researchers to modify the tropism and biodistribution profile of the vector, leading to the production of viral vectors able to target different tissues and organs. This review describes recent non-genetic modifications of the surfaces of viral vectors to decrease immune system activation and to control tissue targeting. The developments described herein provide opportunities for applications of gene therapy to treat acquired disorders and genetic diseases and to become useful tools in regenerative medicine.

  6. Covariantized vector Galileons

    NASA Astrophysics Data System (ADS)

    Hull, Matthew; Koyama, Kazuya; Tasinato, Gianmassimo

    2016-03-01

    Vector Galileons are ghost-free systems containing higher derivative interactions of vector fields. They break the vector gauge symmetry, and the dynamics of the longitudinal vector polarizations acquire a Galileon symmetry in an appropriate decoupling limit in Minkowski space. Using an Arnowitt-Deser-Misner approach, we carefully reconsider the coupling with gravity of vector Galileons, with the aim of studying the necessary conditions to avoid the propagation of ghosts. We develop arguments that put on a more solid footing the results previously obtained in the literature. Moreover, working in analogy with the scalar counterpart, we find indications for the existence of a "beyond Horndeski" theory involving vector degrees of freedom that avoids the propagation of ghosts thanks to secondary constraints. In addition, we analyze a Higgs mechanism for generating vector Galileons through spontaneous symmetry breaking, and we present its consistent covariantization.

  7. Towards Resiliency Evaluation of Vector Programs

    SciTech Connect

    Sharma, Vishal C.; Gopalakrishnan, Ganesh; Krishnamoorthy, Sriram

    2016-08-04

    The systems resilience research community has developed methods to manually insert additional source-program level assertions to trap errors, and also devised tools to conduct fault injection studies for scalar program codes. In this work, we contribute the first vector oriented LLVM-level fault injector VULFI to help study the effects of faults in vector architectures that are of growing importance, especially for vectorizing loops. Using VULFI, we conduct a resiliency study of nine real-world vector benchmarks using Intel’s AVX and SSE extensions as the target vector instruction sets, and offer the first reported understanding of how faults affect vector instruction sets. We take this work further toward automating the insertion of resilience assertions during compilation. This is based on our observation that during intermediate (e.g., LLVM-level) code generation to handle full and partial vectorization, modern compilers exploit (and explicate in their code-documentation) critical invariants. These invariants are turned into error-checking code. We confirm the efficacy of these automatically inserted low-overhead error detectors for vectorized for-loops.

  8. Acute toxicity and repellent activity of the Origanum scabrum Boiss. & Heldr. (Lamiaceae) essential oil against four mosquito vectors of public health importance and its biosafety on non-target aquatic organisms.

    PubMed

    Govindarajan, Marimuthu; Kadaikunnan, Shine; Alharbi, Naiyf S; Benelli, Giovanni

    2016-11-01

    The recent outbreaks of dengue, chikungunya, and Zika virus highlighted the pivotal importance of mosquito vector control in tropical and subtropical areas worldwide. However, mosquito control is facing hot challenges, mainly due to the rapid development of pesticide resistance in Culicidae and the limited success of biocontrol programs on Aedes mosquitoes. In this framework, screening botanicals for their mosquitocidal potential may offer effective and eco-friendly tools in the fight against mosquitoes. In the present study, the essential oil (EO) obtained from the medicinal plant Origanum scabrum was analyzed by GC-MS and evaluated for its mosquitocidal and repellent activities towards Anopheles stephensi, Aedes aegypti, Culex quinquefasciatus, and Culex tritaeniorhynchus. GC-MS analysis showed a total of 28 compounds, representing 97.1 % of the EO. The major constituents were carvacrol (48.2 %) and thymol (16.6 %). The EO was toxic effect to the A. stephensi, A. aegypti, C. quinquefasciatus, and C. tritaeniorhynchus larvae, with LC50 of 61.65, 67.13, 72.45, and 78.87 μg/ml, respectively. Complete ovicidal activity was observed at 160, 200, 240, and 280 μg/ml, respectively. Against adult mosquitoes, LD50 were 122.38, 134.39, 144.53, and 158.87 μg/ml, respectively. In repellency assays, the EOs tested at 1.0, 2.5, and 5.0 mg/cm(2) concentration of O. scabrum gave 100 % protection from mosquito bites up to 210, 180, 150, and 120 min, respectively. From an eco-toxicological point of view, the EO was tested on three non-target mosquito predators, Gambusia affinis, Diplonychus indicus, and Anisops bouvieri, with LC50 ranging from 4162 to 12,425 μg/ml. Overall, the EO from O. scabrum may be considered as a low-cost and eco-friendly source of phytochemicals to develop novel repellents against Culicidae.

  9. Disease Vector Ecology Profile: Haiti

    DTIC Science & Technology

    1994-09-01

    The elderly and children are most susceptible to infection. VECTOR TRANSMISSION: Primary Vectors: Culex nigripalpus , Aedes taeniorhynchus VECTOR...BIONOMICS: Culex nigripalpus breeds in a broad variety of aquatic habitats including lakes, temporary pools, epiphytic plants, brackish water, and...disease. VECTOR TRANSMISSION: Primary Vectors: Culex quinquefasciatus and Cx. nigripalpus ; both species are primary vectors in the U.S., and both

  10. Index Sets and Vectorization

    SciTech Connect

    Keasler, J A

    2012-03-27

    Vectorization is data parallelism (SIMD, SIMT, etc.) - extension of ISA enabling the same instruction to be performed on multiple data items simultaeously. Many/most CPUs support vectorization in some form. Vectorization is difficult to enable, but can yield large efficiency gains. Extra programmer effort is required because: (1) not all algorithms can be vectorized (regular algorithm structure and fine-grain parallelism must be used); (2) most CPUs have data alignment restrictions for load/store operations (obey or risk incorrect code); (3) special directives are often needed to enable vectorization; and (4) vector instructions are architecture-specific. Vectorization is the best way to optimize for power and performance due to reduced clock cycles. When data is organized properly, a vector load instruction (i.e. movaps) can replace 'normal' load instructions (i.e. movsd). Vector operations can potentially have a smaller footprint in the instruction cache when fewer instructions need to be executed. Hybrid index sets insulate users from architecture specific details. We have applied hybrid index sets to achieve optimal vectorization. We can extend this concept to handle other programming models.

  11. Lentiviral vectors in cancer immunotherapy.

    PubMed

    Oldham, Robyn Aa; Berinstein, Elliot M; Medin, Jeffrey A

    2015-01-01

    Basic science advances in cancer immunotherapy have resulted in various treatments that have recently shown success in the clinic. Many of these therapies require the insertion of genes into cells to directly kill them or to redirect the host's cells to induce potent immune responses. Other analogous therapies work by modifying effector cells for improved targeting and enhanced killing of tumor cells. Initial studies done using γ-retroviruses were promising, but safety concerns centered on the potential for insertional mutagenesis have highlighted the desire to develop other options for gene delivery. Lentiviral vectors (LVs) have been identified as potentially more effective and safer alternative delivery vehicles. LVs are now in use in clinical trials for many different types of inherited and acquired disorders, including cancer. This review will discuss current knowledge of LVs and the applications of this viral vector-based delivery vehicle to cancer immunotherapy.

  12. Broad patterns in domestic vector-borne Trypanosoma cruzi transmission dynamics: synanthropic animals and vector control.

    PubMed

    Peterson, Jennifer K; Bartsch, Sarah M; Lee, Bruce Y; Dobson, Andrew P

    2015-10-22

    Chagas disease (caused by Trypanosoma cruzi) is the most important neglected tropical disease (NTD) in Latin America, infecting an estimated 5.7 million people in the 21 countries where it is endemic. It is one of the NTDs targeted for control and elimination by the 2020 London Declaration goals, with the first goal being to interrupt intra-domiciliary vector-borne T. cruzi transmission. A key question in domestic T. cruzi transmission is the role that synanthropic animals play in T. cruzi transmission to humans. Here, we ask, (1) do synanthropic animals need to be targeted in Chagas disease prevention policies?, and (2) how does the presence of animals affect the efficacy of vector control? We developed a simple mathematical model to simulate domestic vector-borne T. cruzi transmission and to specifically examine the interaction between the presence of synanthropic animals and effects of vector control. We used the model to explore how the interactions between triatomine bugs, humans and animals impact the number and proportion of T. cruzi-infected bugs and humans. We then examined how T. cruzi dynamics change when control measures targeting vector abundance are introduced into the system. We found that the presence of synanthropic animals slows the speed of T. cruzi transmission to humans, and increases the sensitivity of T. cruzi transmission dynamics to vector control measures at comparable triatomine carrying capacities. However, T. cruzi transmission is amplified when triatomine carrying capacity increases with the abundance of syntathoropic hosts. Our results suggest that in domestic T. cruzi transmission scenarios where no vector control measures are in place, a reduction in synanthropic animals may slow T. cruzi transmission to humans, but it would not completely eliminate transmission. To reach the 2020 goal of interrupting intra-domiciliary T. cruzi transmission, it is critical to target vector populations. Additionally, where vector control measures

  13. Molecular neurosurgery: vectors and vector delivery strategies.

    PubMed

    White, Edward

    2012-12-01

    Molecular neurosurgery involves the use of vector-mediated gene therapy and gene knockdown to manipulate in vivo gene expression for the treatment of neurological diseases. These techniques have the potential to revolutionise the practice of neurosurgery. However, significant challenges remain to be overcome before these techniques enter routine clinical practice. These challenges have been the subject of intensive research in recent years and include the development of strategies to facilitate effective vector delivery to the brain and the development of both viral and non-viral vectors that are capable of efficient cell transduction without excessive toxicity. This review provides an update on the practice of molecular neurosurgery with particular focus on the practical neurosurgical aspects of vector delivery to the brain. In addition, an introduction to the key vectors employed in clinical trials and a brief overview of previous gene therapy clinical trials is provided. Finally, key areas for future research aimed at increasing the likelihood of the successful translation of gene therapy into clinical trials are highlighted.

  14. HSV Recombinant Vectors for Gene Therapy

    PubMed Central

    Manservigi, Roberto; Argnani, Rafaela; Marconi, Peggy

    2010-01-01

    The very deep knowledge acquired on the genetics and molecular biology of herpes simplex virus (HSV), has allowed the development of potential replication-competent and replication-defective vectors for several applications in human healthcare. These include delivery and expression of human genes to cells of the nervous systems, selective destruction of cancer cells, prophylaxis against infection with HSV or other infectious diseases, and targeted infection to specific tissues or organs. Replication-defective recombinant vectors are non-toxic gene transfer tools that preserve most of the neurotropic features of wild type HSV-1, particularly the ability to express genes after having established latent infections, and are thus proficient candidates for therapeutic gene transfer settings in neurons. A replication-defective HSV vector for the treatment of pain has recently entered in phase 1 clinical trial. Replication-competent (oncolytic) vectors are becoming a suitable and powerful tool to eradicate brain tumours due to their ability to replicate and spread only within the tumour mass, and have reached phase II/III clinical trials in some cases. The progress in understanding the host immune response induced by the vector is also improving the use of HSV as a vaccine vector against both HSV infection and other pathogens. This review briefly summarizes the obstacle encountered in the delivery of HSV vectors and examines the various strategies developed or proposed to overcome such challenges. PMID:20835362

  15. Intracellular trafficking of hybrid gene delivery vectors.

    PubMed

    Keswani, Rahul K; Lazebnik, Mihael; Pack, Daniel W

    2015-06-10

    Viral and non-viral gene delivery vectors are in development for human gene therapy, but both exhibit disadvantages such as inadequate efficiency, lack of cell-specific targeting or safety concerns. We have recently reported the design of hybrid delivery vectors combining retrovirus-like particles with synthetic polymers or lipids that are efficient, provide sustained gene expression and are more stable compared to native retroviruses. To guide further development of this promising class of gene delivery vectors, we have investigated their mechanisms of intracellular trafficking. Moloney murine leukemia virus-like particles (M-VLPs) were complexed with chitosan (Chi) or liposomes (Lip) comprising DOTAP, DOPE and cholesterol to form the hybrid vectors (Chi/M-VLPs and Lip/M-VLPs, respectively). Transfection efficiency and cellular internalization of the vectors were quantified in the presence of a panel of inhibitors of various endocytic pathways. Intracellular transport and trafficking kinetics of the hybrid vectors were dependent on the synthetic component and used a combination of clathrin- and caveolar-dependent endocytosis and macropinocytosis. Chi/M-VLPs were slower to transfect compared to Lip/M-VLPs due to the delayed detachment of the synthetic component. The synthetic component of hybrid gene delivery vectors plays a significant role in their cellular interactions and processing and is a key parameter for the design of more efficient gene delivery vehicles.

  16. Optimum instantaneous impulsive orbital injection to attain a specified asymptotic velocity vector.

    NASA Technical Reports Server (NTRS)

    Bean, W. C.

    1971-01-01

    A nalysis of the necessary conditions of Battin for instantaneous orbital injection, with consideration of the uniqueness of his solution, and of the further problem which arises in the degenerate case when radius vector and asymptotic vector are separated by 180 deg. It is shown that when the angular separation between radius vector and asymptotic velocity vector satisfies theta not equal to 180 deg, there are precisely two insertion-velocity vectors which permit attainment of the target asymptotic velocity vector, one yielding posigrade, the other retrograde motion. When theta equals to 180 deg, there is a family of insertion-velocity vectors which permit attainment of a specified asymptotic velocity vector with a unique insertion-velocity vector for every arbitrary orientation of a target unit angular momentum vector.

  17. Evaluation of attractive toxic sugar bait (ATSB)-barrier for control of vector and nuisance mosquitoes and its effect on non-target organisms in sub-tropical environments in Florida

    USDA-ARS?s Scientific Manuscript database

    We evaluated the efficacy of attractive toxic sugar baits (ATSB) in the laboratory and the field with the Environmental Protection Agency exempt active ingredient eugenol against vector and nuisance mosquitoes. In the laboratory, eugenol combined in attractive sugar bait (ASB) solution provided high...

  18. Design and Evaluation of RGD-Modified Gemini Surfactant-Based Lipoplexes for Targeted Gene Therapy in Melanoma Model.

    PubMed

    Mohammed-Saeid, Waleed; Chitanda, Jackson; Al-Dulaymi, Mays; Verrall, Ronald; Badea, Ildiko

    2017-09-01

    We have developed and evaluated novel peptide-targeted gemini surfactant-based lipoplexes designed for melanoma gene therapy. Integrin receptor targeting peptide, cyclic-arginylglycylaspartic acid (cRGD), was either chemically coupled to a gemini surfactant backbone or physically co-formulated with lipoplexes. Several formulations and transfection techniques were developed. Transfection efficiency and cellular toxicity of the lipoplexes were evaluated in an in vitro human melanoma model. Physicochemical properties were examined using dynamic light scattering, zeta-potential, and small-angle X-ray scattering measurements. RGD-modified gemini surfactant based lipoplexes showed significant enhancement in gene transfection activity in A375 cell lines compared to the standard non-targeted formulation, especially when RGD was chemically conjugated to the gemini surfactant (RGD-G). The RGD had no effect on the cell toxicity profile of the lipoplex systems. Targeting specificity was confirmed by using an excess of free RGD and negative control peptide (RAD) and was demonstrated by using normal human epidermal keratinocytes. Physicochemical characterization showed that all nanoparticles were in the optimal size range for cellular uptake and there were no significant differences between RGD-modified and standard lipoplexes. These findings indicate the potential of RGD-modified gemini surfactant-based lipoplexes for use in melanoma gene therapy as an alternative to conventional chemotherapy.

  19. Effective reduction of nonspecific binding by surface engineering of quantum dots with bovine serum albumin for cell-targeted imaging.

    PubMed

    Zhang, Bingbo; Wang, Xiaohui; Liu, Fengjun; Cheng, Yingsheng; Shi, Donglu

    2012-12-04

    Quantum dots (QDs) have been widely used as fluorescent probes in cell-targeted imaging. However, nonspecific binding to cellular membranes has been a major challenge. In this study, a new approach is developed for effective reduction of nonspecific binding by bovine serum albumin (BSA)-coated QDs in cell targeting. The experimental results show efficient transfer of hydrophobic QDs from organic to aqueous phase in the presence of BSA aqueous solution under ultrasonication. This ultrasonication-based approach is facile, rapid, and efficient. Stabilization of QDs is mainly achieved by multiple mercapto groups in BSA macromolecules as multidentate ligands and partially by hydrophobic interaction between BSA and pending fatty ligands on QDs. The water solubility of QDs is enhanced by the surface amino and carboxyl groups, which also provide reaction sites for conjugation of targeting ligands. The BSA-coated QDs, with an overwhelming majority of hydrodynamic diameter size of ca. 18 nm, are colloidally stable under both acidic and basic conditions and found to exhibit strong fluorescent intensities. The nonspecific cellular binding is effectively reduced by BSA-coated QDs, compared with the mercaptopropionic acid (MPA)-coated CdTe QDs. BSA-coated QDs are further functionalized with cyclic Arg-Gly-Asp (cRGD) peptide. The cell assays indicate their high target-selectivity in integrin α(v)β(3)-expressed cell imaging.

  20. Paramagnetic hollow silica nanospheres for in vivo targeted ultrasound and magnetic resonance imaging.

    PubMed

    An, Lu; Hu, He; Du, Jing; Wei, Jie; Wang, Li; Yang, Hong; Wu, Dongmei; Shi, Haili; Li, Fenghua; Yang, Shiping

    2014-07-01

    A series of hollow silica nanospheres (HSNSs) with sizes ranging from 100 to 400 nm were synthesized and used for primary ultrasound imaging (US) efficiency assessment. The 400 nm HSNSs were chosen as platform for conjugation with Gd-DTPA and cyclo-arginine-glycine-aspartic acid c(RGD) peptide to construct US and magnetic resonance imaging (MRI) dual-modal contrast agents (CAs): [HSNSs@(DTPA-Gd)-RGD]. The obtained CAs displayed good physiological stability, low cytotoxicity and negligible hemolytic activity in vitro. Furthermore, the passive accumulation and active-targeting of the HSNSs in the tumor site of mice was demonstrated by US and MR imaging, respectively. The qualitative and quantitative biodistribution of the HSNSs showed that they mainly accumulated in the tissues of liver, lung, tumor after intravenous administration and then be excreted from feces. In addition, histological, hematological, blood and biochemical analysis were used to further study toxicity of the HSNSs, and all results indicated that there were no covert toxicity of HSNSs in mice after long exposure times. Findings from this study indicated that the silica-based paramagnetic HSNSs can be used as a platform for long-term targeted imaging and therapy studies safely in vivo. Copyright © 2014 Elsevier Ltd. All rights reserved.

  1. Construction and expression of recombined human AFP eukaryotic expression vector

    PubMed Central

    Zhang, Li-Wang; Ren, Jun; Zhang, Liang; Zhang, Hong-Mei; Jin, Bin; Pan, Bo-Rong; Si, Xiao-Ming; Zhang, Yan-Jun; Wang, Zhong-Hua; Pan, Yang-Lin; Festein, Stephen M

    2003-01-01

    AIM: To construct a recombined human AFP eukaryotic expression vector for the purpose of gene therapy and target therapy of hepatocellular carcinoma (HCC). METHODS: The full length AFP-cDNA of prokaryotic vector was digested, and subcloned to the multi-clony sites of the eukaryotic vector. The constructed vector was confirmed by enzymes digestion and electrophoresis, and the product expressed was detected by electrochemiluminescence and immunofluorescence methods. RESULTS: The full length AFP-cDNA successfully cloned to the eukaryotic vector through electrophoresis, 0.9723 IU/mL AFP antigen was detected in the supernatant of AFP-CHO by electrochemiluminescence method. Compared with the control groups, the differences were significant (P < 0.05). AFP antigen molecule was observed in the plasma of AFP-CHO by immunofluorescence staining. CONCLUSION: The recombined human AFP eukaryotic expression vector can express in CHO cell line. It provides experimental data for gene therapy and target therapy of hepatocellular carcinoma. PMID:12854142

  2. Vector generator scan converter

    DOEpatents

    Moore, James M.; Leighton, James F.

    1990-01-01

    High printing speeds for graphics data are achieved with a laser printer by transmitting compressed graphics data from a main processor over an I/O (input/output) channel to a vector generator scan converter which reconstructs a full graphics image for input to the laser printer through a raster data input port. The vector generator scan converter includes a microprocessor with associated microcode memory containing a microcode instruction set, a working memory for storing compressed data, vector generator hardward for drawing a full graphic image from vector parameters calculated by the microprocessor, image buffer memory for storing the reconstructed graphics image and an output scanner for reading the graphics image data and inputting the data to the printer. The vector generator scan converter eliminates the bottleneck created by the I/O channel for transmitting graphics data from the main processor to the laser printer, and increases printer speed up to thirty fold.

  3. Vector generator scan converter

    DOEpatents

    Moore, J.M.; Leighton, J.F.

    1988-02-05

    High printing speeds for graphics data are achieved with a laser printer by transmitting compressed graphics data from a main processor over an I/O channel to a vector generator scan converter which reconstructs a full graphics image for input to the laser printer through a raster data input port. The vector generator scan converter includes a microprocessor with associated microcode memory containing a microcode instruction set, a working memory for storing compressed data, vector generator hardware for drawing a full graphic image from vector parameters calculated by the microprocessor, image buffer memory for storing the reconstructed graphics image and an output scanner for reading the graphics image data and inputting the data to the printer. The vector generator scan converter eliminates the bottleneck created by the I/O channel for transmitting graphics data from the main processor to the laser printer, and increases printer speed up to thirty fold. 7 figs.

  4. An Oncotropic Adenovirus Vector System for Breast Cancer Treatment

    DTIC Science & Technology

    2005-09-01

    AD Award Number: DAMD17-03-1-0629 TITLE: An Oncotropic Adenovirus Vector System for Breast Cancer Treatment PRINCIPAL INVESTIGATOR: Igor P. Dmitriev...Aug 2005 4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER An Oncotropic Adenovirus Vector System for Breast Cancer Treatment 5b. GRANT NUMBER DAMD17-03-1...epithelial cells, the origin of most human cancers. However, realization of the full potential of Ad vectors for targeted cancer treatment is currently

  5. Line Integral of a Vector.

    ERIC Educational Resources Information Center

    Balabanian, Norman

    This programed booklet is designed for the engineering student who understands and can use vector and unit vector notation, components of a vector, parallel law of vector addition, and the dot product of two vectors. Content begins with work done by a force in moving a body a certain distance along some path. For each of the examples and problem…

  6. Cyclic RGD peptide-modified liposomal drug delivery system for targeted oral apatinib administration: enhanced cellular uptake and improved therapeutic effects

    PubMed Central

    Song, Zhiwang; Lin, Yun; Zhang, Xia; Feng, Chan; Lu, Yonglin; Gao, Yong; Dong, Chunyan

    2017-01-01

    Apatinib is an oral tyrosine kinase inhibitor, which selectively targets vascular endothelial growth factor receptor 2 and has the potential to treat many tumors therapeutically. Cyclic arginylglycylaspartic acid (cRGD)- and polyethylene glycol (PEG)-modified liposomes (cRGD-Lipo-PEG) were constructed to act as a targeted delivery system for the delivery of apatinib to the human colonic cancer cell line, HCT116. These cRGD-modified liposomes specifically recognized integrin αvβ3 and exhibited greater uptake efficiency with respect to delivering liposomes into HCT116 cells when compared to nontargeted liposomes (Lipo-PEG), as well as greater death of tumor cells and apoptosis. The mechanism by which cRGD-Lipo-PEG targets cells was elucidated further with competition assays. To determine the anticancer efficacy in vivo, nude mice were implanted with HCT116 xenografts and treated with apatinib-loaded liposomes or free apatinib intravenously or via intragastric administration. The active and passive targeting of cRGD-Lipo-PEG led to significant tumor treatment targeting ability, better inhibition of tumor growth, and less toxicity when compared with treatments using uncombined apatinib. The results presented strongly support the case for cRGD-Lipo-PEG representing a targeted delivery system for apatinib in the treatment of colonic cancer. PMID:28331317

  7. Viral vectors and delivery strategies for CNS gene therapy

    PubMed Central

    Gray, Steven J; Woodard, Kenton T; Samulski, R Jude

    2015-01-01

    This review aims to provide a broad overview of the targets, challenges and potential for gene therapy in the CNS, citing specific examples. There are a broad range of therapeutic targets, with very different requirements for a suitable viral vector. By utilizing different vector tropisms, novel routes of administration and engineered promoter control, transgenes can be targeted to specific therapeutic applications. Viral vectors have proven efficacious in preclinical models for several disease applications, spurring several clinical trials. While the field has pushed the limits of existing adeno-associated virus-based vectors, a next generation of vectors based on rational engineering of viral capsids should expand the application of gene therapy to be more effective in specific therapeutic applications. PMID:22833965

  8. Baculovirus Transfer Vectors.

    PubMed

    Possee, Robert D; King, Linda A

    2016-01-01

    The production of a recombinant baculovirus expression vector normally involves mixing infectious virus DNA with a plasmid-based transfer vector and then co-transfecting insect cells to initiate virus infection. The aim of this chapter is to provide an update on the range of baculovirus transfer vectors currently available. Some of the original transfer vectors developed are now difficult to obtain but generally have been replaced by superior reagents. We focus on those that are available commercially and should be easy to locate. These vectors permit the insertion of single or multiple genes for expression, or the production of proteins with specific peptide tags that aid subsequent protein purification. Others have signal peptide coding regions permitting protein secretion or plasma membrane localization. A table listing the transfer vectors also includes information on the parental virus that should be used with each one. Methods are described for the direct insertion of a recombinant gene into the virus genome without the requirement for a transfer vector. The information provided should enable new users of the system to choose those reagents most suitable for their purposes.

  9. Stimulation of IgY responses in gene gun immunized laying hens by combined administration of vector DNA coding for the target antigen Botulinum toxin A1 and for avian cytokine adjuvants.

    PubMed

    Niederstadt, Lars; Hohn, Oliver; Dorner, Brigitte G; Schade, Rüdiger; Bannert, Norbert

    2012-08-31

    DNA immunization is a convenient and effective way of inducing a specific antibody response. In mammals, co-administration of vectors encoding immunostimulatory cytokines can enhance the humoral response resulting in elevated antibody titers. We therefore set out to investigate the effect using avian interleukin 1β (IL-1β) and avian interleukin 6 (IL-6) as genetic adjuvants when immunizing laying hens. A BoNT A1 holotoxoid DNA immunogen carrying two inactivating mutations was evaluated for its ability to induce a specific and sustained IgY antibody response. Both the holotoxoid and the cytokine sequences were codon-optimized. In vitro, the proteins were efficiently expressed in transfected HEK 293T cells and the cytokines were secreted into the culture supernatants. Whereas eggs from hens immunized via gene gun using a prime boost strategy showed no differences in their total IgY content, the specific αBoNT A1 response was slightly elevated up to 1.4× by the IL-1β adjuvant vector and increased by 3.8× by the IL-6 vector. Finally, although hens receiving the IL-1β adjuvant had laying capacities above the average, hens receiving the IL-6 adjuvant experienced laying problems.

  10. Null Killing vectors

    NASA Astrophysics Data System (ADS)

    Lukács, B.; Perjés, Z.; Sebestyén, Á.

    1981-06-01

    Space-times admitting a null Killing vector are studied, using the Newman-Penrose spin coefficient formalism. The properties of the eigenrays (principal null curves of the Killing bivector) are shown to be related to the twist of the null Killing vector. Among the electrovacs, the ones containing a null Maxwell field turn out to belong to the twist-free class. An electrovac solution is obtained for which the null Killing vector is twisting and has geodesic and shear-free eigenrays. This solution is parameterless and appears to be the field of a zero-mass, spinning, and charged source.

  11. Mechanisms of cell penetration and cytotoxicity of ultrasmall Au nanoparticles conjugated to doxorubicin and/or targeting peptides

    NASA Astrophysics Data System (ADS)

    Nadeau, Jay; Poon, Wilson; Zhang, Xuan

    2015-03-01

    The goals of this work were to determine whether conjugation of any of four selected peptides to Au nanoparticles improved their delivery to B16 melanoma in vitro and in vivo. In in vitro cytotoxicity assays, peptides and conjugates were endocytosed but did not escape from endosomes. None of the peptides showed any cytotoxicity, with or without conjugation to the nanoparticles. The combination of peptides and doxorubicin did not improve upon the cytotoxicity of gold-doxorubicin alone. We then tested targeting in vivo using inductively coupled plasma mass spectrometry to quantify the concentration of Au in the organs of B16 tumor-bearing mice 4, 24, and 72 h after intravenous Au nanoparticle injection. These experiments showed that in some cases, peptide conjugation improved upon the enhanced permeability and retention (EPR) effect. A peptide based upon the myxoma virus and the cyclic RGD peptide were both effective at tumor targeting; myxoma was more effective with un-PEGylated particles, and cRGD with PEGylated particles. The FREG and melanocyte stimulating hormone (MSH) peptides did not improve targeting. These results suggest that these peptides may improve delivery of Au particles to tumors, but also may prevent entry of particles into cell nuclei.

  12. cRGD-Modified Benzimidazole-based pH-Responsive Nanoparticles for Enhanced Tumor Targeted Doxorubicin Delivery.

    PubMed

    Liu, Jinjian; Liu, Qian; Yang, Cuihong; Sun, Yu; Zhang, Yumin; Huang, Pingsheng; Zhou, Junhui; Liu, Qiang; Chu, Liping; Huang, Fan; Deng, Liandong; Dong, Anjie; Liu, Jianfeng

    2016-05-04

    Finding a smart cancer drug delivery carrier with long blood circulation, enhanced cancer targeting, and quick drug release in tumors is critical for efficient cancer chemotherapy. Herein, we design a cRGD-polycarboxybetaine methacrylate-b-polybenzimidazole methacrylate (cRGD-PCB-b-PBBMZ) copolymer to self-assemble into smart drug-loaded nanoparticles (cRGD-PCM NPs) which can target αvβ3 integrin overexpressed cancer tissue by cRGD peptide unit and release drug quickly in cancer cells by protonation of benzimidazole groups. The outer PCB layer can resist protein adhesion, and there are only about 10% of proteins in mouse serum adhered to the surface of PCM NPs. With the pKa value of 5.08 of the benzimidazole units, DOX can be released from NPs in pH 5.0 PBS. cRGD-PCM NPs can bring more DOX into HepG2 cells than nontargeting PCM NPs, and there has high DOX release rate in HepG2 cells because of the protonation of benzimidazole groups in endosome and lysosome. MTT assay verifies that higher cellular uptake of DOX causes higher cytotoxicity. Furthermore, the results of ex vivo imaging studies confirm that cRGD-PCM/DOX NPs can successfully deliver DOX into tumor tissue from the injection site. Therefore, the multifunctional cRGD-PCM NPs show great potential as novel nanocarriers for targeting cancer chemotherapy.

  13. Motor planning poststroke: impairment in vector-coded reach plans.

    PubMed

    Rizzo, John-Ross; Hudson, Todd E; Abdou, Andrew; Rashbaum, Ira G; George, Ajax E; Raghavan, Preeti; Landy, Michael S

    2015-12-01

    Healthy individuals appear to use both vector-coded reach plans that encode movements in terms of their desired direction and extent, and target-coded reach plans that encode the desired endpoint position of the effector. We examined whether these vector and target reach-planning codes are differentially affected after stroke. Participants with stroke and healthy controls made blocks of reaches that were grouped by target location (providing target-specific practice) and by movement vector (providing vector-specific practice). Reach accuracy was impaired in the more affected arm after stroke, but not distinguishable for target- versus vector-grouped reaches. Reach velocity and acceleration were not only impaired in both the less and more affected arms poststroke, but also not distinguishable for target- versus vector-grouped reaches. As previously reported in controls, target-grouped reaches yielded isotropic (circular) error distributions and vector-grouped reaches yielded error distributions elongated in the direction of the reach. In stroke, the pattern of variability was similar. However, the more affected arm showed less elongated error ellipses for vector-grouped reaches compared to the less affected arm, particularly in individuals with right-hemispheric stroke. The results suggest greater impairment to the vector-coded movement-planning system after stroke, and have implications for the development of personalized approaches to poststroke rehabilitation: Motor learning may be enhanced by practice that uses the preserved code or, conversely, by retraining the more impaired code to restore function. © 2015 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.

  14. The Vector Decomposition Problem

    NASA Astrophysics Data System (ADS)

    Yoshida, Maki; Mitsunari, Shigeo; Fujiwara, Toru

    This paper introduces a new computational problem on a two-dimensional vector space, called the vector decomposition problem (VDP), which is mainly defined for designing cryptosystems using pairings on elliptic curves. We first show a relation between the VDP and the computational Diffie-Hellman problem (CDH). Specifically, we present a sufficient condition for the VDP on a two-dimensional vector space to be at least as hard as the CDH on a one-dimensional subspace. We also present a sufficient condition for the VDP with a fixed basis to have a trapdoor. We then give an example of vector spaces which satisfy both sufficient conditions and on which the CDH is assumed to be hard in previous work. In this sense, the intractability of the VDP is a reasonable assumption as that of the CDH.

  15. Vector inflation and vortices

    SciTech Connect

    Lewis, C.M. )

    1991-09-15

    A vector field {ital A}{sub {mu}} is coupled to the Einstein equations with a linearly perturbed Friedmann-Robertson-Walker metric, constructed to generate first-order vector perturbations. A working classical chaotic vector inflation is demonstrated and then quantum fluctuations of the field are used to constrain the cosmological perturbations. In particular, the vector momentum flux {ital T}{sub 0{ital i}} is tracked to the epoch where radiation-dominated matter exists. Matching conditions using observational constraints of the cosmic microwave background radiation give rise to a peculiar cosmological velocity of the order of 10{sup {minus}100}{ital c}. Amplification of this number, e.g., by breaking the conformal invariance of the field, could be used to generate cosmic magnetic fields using a dynamo mechanism.

  16. Saccharomyces cerevisiae Shuttle vectors.

    PubMed

    Gnügge, Robert; Rudolf, Fabian

    2017-01-10

    Yeast shuttle vectors are indispensable tools in yeast research. They enable cloning of defined DNA sequences in Escherichia coli and their direct transfer into Saccharomyces cerevisiae cells. There are three types of commonly used yeast shuttle vectors: centromeric plasmids, episomal plasmids and integrating plasmids. In this review, we discuss the different plasmid systems and their characteristic features. We focus on their segregational stability and copy number and indicate how to modify these properties. Copyright © 2017 John Wiley & Sons, Ltd.

  17. Poynting-vector filter

    DOEpatents

    Carrigan, Charles R [Tracy, CA

    2011-08-02

    A determination is made of frequency components associated with a particular bearing or location resulting from sources emitting electromagnetic-wave energy for which a Poynting-Vector can be defined. The broadband frequency components associated with a specific direction or location of interest are isolated from other components in the power spectrum that are not associated with the direction or location of interest. The collection of pointing vectors can be used to characterize the source.

  18. Bloch vector projection noise

    NASA Technical Reports Server (NTRS)

    Wang, Li-Jun; Bacon, A. M.; Zhao, H.-Z.; Thomas, J. E.

    1994-01-01

    In the optical measurement of the Bloch vector components describing a system of N two-level atoms, the quantum fluctuations in these components are coupled into the measuring optical field. This paper develops the quantum theory of optical measurement of Bloch vector projection noise. The preparation and probing of coherence in an effective two-level system consisting of the two ground states in an atomic three-level lambda-scheme are analyzed.

  19. Emergence and prevalence of human vector-borne diseases in sink vector populations.

    PubMed

    Rascalou, Guilhem; Pontier, Dominique; Menu, Frédéric; Gourbière, Sébastien

    2012-01-01

    Vector-borne diseases represent a major public health concern in most tropical and subtropical areas, and an emerging threat for more developed countries. Our understanding of the ecology, evolution and control of these diseases relies predominantly on theory and data on pathogen transmission in large self-sustaining 'source' populations of vectors representative of highly endemic areas. However, there are numerous places where environmental conditions are less favourable to vector populations, but where immigration allows them to persist. We built an epidemiological model to investigate the dynamics of six major human vector borne-diseases in such non self-sustaining 'sink' vector populations. The model was parameterized through a review of the literature, and we performed extensive sensitivity analysis to look at the emergence and prevalence of the pathogen that could be encountered in these populations. Despite the low vector abundance in typical sink populations, all six human diseases were able to spread in 15-55% of cases after accidental introduction. The rate of spread was much more strongly influenced by vector longevity, immigration and feeding rates, than by transmission and virulence of the pathogen. Prevalence in humans remained lower than 5% for dengue, leishmaniasis and Japanese encephalitis, but substantially higher for diseases with longer duration of infection; malaria and the American and African trypanosomiasis. Vector-related parameters were again the key factors, although their influence was lower than on pathogen emergence. Our results emphasize the need for ecology and evolution to be thought in the context of metapopulations made of a mosaic of sink and source habitats, and to design vector control program not only targeting areas of high vector density, but working at a larger spatial scale.

  20. Emergence and Prevalence of Human Vector-Borne Diseases in Sink Vector Populations

    PubMed Central

    Rascalou, Guilhem; Pontier, Dominique; Menu, Frédéric; Gourbière, Sébastien

    2012-01-01

    Vector-borne diseases represent a major public health concern in most tropical and subtropical areas, and an emerging threat for more developed countries. Our understanding of the ecology, evolution and control of these diseases relies predominantly on theory and data on pathogen transmission in large self-sustaining ‘source’ populations of vectors representative of highly endemic areas. However, there are numerous places where environmental conditions are less favourable to vector populations, but where immigration allows them to persist. We built an epidemiological model to investigate the dynamics of six major human vector borne-diseases in such non self-sustaining ‘sink’ vector populations. The model was parameterized through a review of the literature, and we performed extensive sensitivity analysis to look at the emergence and prevalence of the pathogen that could be encountered in these populations. Despite the low vector abundance in typical sink populations, all six human diseases were able to spread in 15–55% of cases after accidental introduction. The rate of spread was much more strongly influenced by vector longevity, immigration and feeding rates, than by transmission and virulence of the pathogen. Prevalence in humans remained lower than 5% for dengue, leishmaniasis and Japanese encephalitis, but substantially higher for diseases with longer duration of infection; malaria and the American and African trypanosomiasis. Vector-related parameters were again the key factors, although their influence was lower than on pathogen emergence. Our results emphasize the need for ecology and evolution to be thought in the context of metapopulations made of a mosaic of sink and source habitats, and to design vector control program not only targeting areas of high vector density, but working at a larger spatial scale. PMID:22629337

  1. Light Vector Mesons in the Nuclear Medium

    SciTech Connect

    Wood, Michael; Nasseripour, Rakhsha; Weygand, Dennis; Djalali, Chaden; Tur, Clarisse; Mosel, Ulrich; Muehlich, Pascal; Adams, Gary; Amaryan, Moscov; Amaryan, Moskov; Ambrozewicz, Pawel; Anghinolfi, Marco; Asryan, Gegham; Avagyan, Harutyun; Baghdasaryan, Hovhannes; Baillie, Nathan; Ball, James; Baltzell, Nathan; Barrow, Steve; Battaglieri, Marco; Bedlinskiy, Ivan; Bektasoglu, Mehmet; Bellis, Matthew; Benmouna, Nawal; Berman, Barry; Biselli, Angela; Blaszczyk, Lukasz; Bouchigny, Sylvain; Boyarinov, Sergey; Bradford, Robert; Branford, Derek; Briscoe, William; Brooks, William; Burkert, Volker; Butuceanu, Cornel; Calarco, John; Careccia, Sharon; Carman, Daniel; Carnahan, Bryan; Casey, Liam; Chen, Shifeng; Cheng, Lu; Cole, Philip; Collins, Patrick; Coltharp, Philip; Crabb, Donald; Crannell, Hall; Crede, Volker; Cummings, John; Dashyan, Natalya; De Vita, Raffaella; De Sanctis, Enzo; Degtiarenko, Pavel; Denizli, Haluk; Dennis, Lawrence; Deur, Alexandre; Dharmawardane, Kahanawita; Dickson, Richard; Dodge, Gail; Doughty, David; Dugger, Michael; Dytman, Steven; Dzyubak, Oleksandr; Egiyan, Hovanes; Egiyan, Kim; Elfassi, Lamiaa; Elouadrhiri, Latifa; Eugenio, Paul; Fedotov, Gleb; Feldman, Gerald; Feuerbach, Robert; Fradi, Ahmed; Funsten, Herbert; Garcon, Michel; Gavalian, Gagik; Gilfoyle, Gerard; Giovanetti, Kevin; Girod, Francois-Xavier; Goetz, John; Gordon, Christopher; Gothe, Ralf; Griffioen, Keith; Guidal, Michel; Guler, Nevzat; Guo, Lei; Gyurjyan, Vardan; Hadjidakis, Cynthia; Hafidi, Kawtar; Hakobyan, Hayk; Hakobyan, Rafael; Hanretty, Charles; Hardie, John; Hassall, Neil; Hersman, F.; Hicks, Kenneth; Hleiqawi, Ishaq; Holtrop, Maurik; Hyde, Charles; Ilieva, Yordanka; Ireland, David; Ishkhanov, Boris; Isupov, Evgeny; Ito, Mark; Jenkins, David; Jo, Hyon-Suk; Johnstone, John; Joo, Kyungseon; Juengst, Henry; Kalantarians, Narbe; Kellie, James; Khandaker, Mahbubul; Khetarpal, Puneet; Kim, Wooyoung; Klein, Andreas; Klein, Franz; Klimenko, Alexei; Kossov, Mikhail; Krahn, Zebulun; Kramer, Laird; Kubarovsky, Valery; Kuhn, Joachim; Kuhn, Sebastian; Kuleshov, Sergey; Lachniet, Jeff; Laget, Jean; Langheinrich, Jorn; Lawrence, David; Li, Ji; Livingston, Kenneth; Lu, Haiyun; MacCormick, Marion; Markov, Nikolai; Mattione, Paul; McAleer, Simeon; McKinnon, Bryan; McNabb, John; Mecking, Bernhard; Mehrabyan, Surik; Melone, Joseph; Mestayer, Mac; Meyer, Curtis; Mibe, Tsutomu; Mikhaylov, Konstantin; Minehart, Ralph; Mirazita, Marco; Miskimen, Rory; Mokeev, Viktor; Moriya, Kei; Morrow, Steven; Moteabbed, Maryam; Mueller, James; Munevar Espitia, Edwin; Mutchler, Gordon; Nadel-Turonski, Pawel; Niccolai, Silvia; Niculescu, Gabriel; Niculescu, Maria-Ioana; Niczyporuk, Bogdan; Niroula, Megh; Niyazov, Rustam; Nozar, Mina; Osipenko, Mikhail; Ostrovidov, Alexander; Park, Kijun; Pasyuk, Evgueni; Paterson, Craig; Pereira, Sergio; Pierce, Joshua; Pivnyuk, Nikolay; Pocanic, Dinko; Pogorelko, Oleg; Pozdnyakov, Sergey; Preedom, Barry; Price, John; Prok, Yelena; Protopopescu, Dan; Raue, Brian; Riccardi, Gregory; Ricco, Giovanni; Ripani, Marco; Ritchie, Barry; Ronchetti, Federico; Rosner, Guenther; Rossi, Patrizia; Sabatie, Franck; Salamanca, Julian; Salgado, Carlos; Santoro, Joseph; Sapunenko, Vladimir; Schumacher, Reinhard; Serov, Vladimir; Sharabian, Youri; Sharov, Dmitri; Shvedunov, Nikolay; Smith, Elton; Smith, Lee; Sober, Daniel; Sokhan, Daria; Stavinsky, Aleksey; Stepanyan, Stepan; Stepanyan, Samuel; Stokes, Burnham; Stoler, Paul; Strakovski, Igor; Strauch, Steffen; Taiuti, Mauro; Tedeschi, David; Tkabladze, Avtandil; Tkachenko, Svyatoslav; Todor, Luminita; Ungaro, Maurizio; Vineyard, Michael; Vlassov, Alexander; Watts, Daniel; Weinstein, Lawrence; Williams, Michael; Wolin, Elliott; Yegneswaran, Amrit; Zana, Lorenzo; Zhang, Bin; Zhang, Jixie; Zhao, Bo; Zhao, Zhiwen

    2008-07-01

    The light vector mesons ($\\rho$, $\\omega$, and $\\phi$) were produced in deuterium, carbon, titanium, and iron targets in a search for possible in-medium modifications to the properties of the $\\rho$ meson at normal nuclear densities and zero temperature. The vector mesons were detected with the CEBAF Large Acceptance Spectrometer (CLAS) via their decays to $e^{+}e^{-}$. The rare leptonic decay was chosen to reduce final-state interactions. A combinatorial background was subtracted from the invariant mass spectra using a well-established event-mixing technique. The $\\rho$ meson mass spectrum was extracted after the $\\omega$ and $\\phi$ signals were removed in a nearly model-independent way. Comparisons were made between the $\\rho$ mass spectra from the heavy targets ($A > 2$) with the mass spectrum extracted from the deuterium target. With respect to the $\\rho$-meson mass, we obtain a small shift compatible with zero. Also, we measure widths consistent with standard nuclear many-body eff

  2. The Inside Out of Lentiviral Vectors

    PubMed Central

    Durand, Stéphanie; Cimarelli, Andrea

    2011-01-01

    Lentiviruses induce a wide variety of pathologies in different animal species. A common feature of the replicative cycle of these viruses is their ability to target non-dividing cells, a property that constitutes an extremely attractive asset in gene therapy. In this review, we shall describe the main basic aspects of the virology of lentiviruses that were exploited to obtain efficient gene transfer vectors. In addition, we shall discuss some of the hurdles that oppose the efficient genetic modification mediated by lentiviral vectors and the strategies that are being developed to circumvent them. PMID:22049307

  3. Syngeneic AAV pseudo-vectors potentiates full vector transduction

    USDA-ARS?s Scientific Manuscript database

    An excessive amount of empty capsids are generated during regular AAV vector production process. These pseudo-vectors often remain in final vectors used for animal studies or clinical trials. The potential effects of these pseudo-vectors on AAV transduction have been a major concern. In the current ...

  4. Packaging of an AAV vector encoding human acid alpha-glucosidase for gene therapy in glycogen storage disease type II with a modified hybrid adenovirus-AAV vector.

    PubMed

    Sun, Baodong; Chen, Y-T; Bird, Andrew; Xu, Fang; Hou, Yang-Xun; Amalfitano, Andrea; Koeberl, Dwight D

    2003-04-01

    We have developed an improved method for packaging adeno-associated virus (AAV) vectors with a replication-defective adenovirus-AAV (Ad-AAV) hybrid virus. The AAV vector encoding human acid alpha-glucosidase (hGAA) was cloned into an E1, polymerase/preterminal protein-deleted adenovirus, such that it is packaged as an Ad vector. Importantly, the Ad-AAV hybrid cannot replicate during AAV vector packaging in 293 cells, because of deletion of polymerase/preterminal protein. The residual Ad-AAV in the AAV vector stock was reduced to <1 infectious particle per 10(10) AAV vector particles. These modifications resulted in approximately 30-fold increased packaging of the AAV vector for the hybrid Ad-AAV vector method as compared with standard transfection-only methods. Similarly improved packaging was demonstrated for pseudotyping the AAV vector as AAV6, and for AAV vector packaging with a second Ad-AAV vector encoding canine glucose-6-phosphatase. Liver-targeted delivery of either the Ad-AAV hybrid or AAV vector particles in acid alpha-glucosidase-knockout (GAA-KO) mice revealed secretion of hGAA with the Ad-AAV vector, and sustained secretion of hGAA with an AAV vector in hGAA-tolerant GAA-KO mice. Further development of hybrid Ad-AAV vectors could offer distinct advantages for gene therapy in glycogen storage diseases.

  5. Boswellia ovalifoliolata (Burseraceae) essential oil as an eco-friendly larvicide? Toxicity against six mosquito vectors of public health importance, non-target mosquito fishes, backswimmers, and water bugs.

    PubMed

    Benelli, Giovanni; Rajeswary, Mohan; Vijayan, Periasamy; Senthilmurugan, Sengamalai; Alharbi, Naiyf S; Kadaikunnan, Shine; Khaled, Jamal M; Govindarajan, Marimuthu

    2017-03-22

    The use of synthetic pesticides to control vector populations is detrimental to human health and the environment and may lead to the development of resistant strains. Plants can be alternative sources of safer compounds effective on mosquito vectors. In this study, the mosquito larvicidal activity of Boswellia ovalifoliolata leaf essential oil (EO) was evaluated against Anopheles stephensi, Anopheles subpictus, Aedes aegypti, Aedes albopictus, Culex quinquefasciatus, and Culex tritaeniorhynchus. GC-MS revealed that the B. ovalifoliolata EO contained at least 20 compounds. The main constituents were β-pinene, α-terpineol, and caryophyllene. In acute toxicity assays, the EO was toxic to larvae of An. stephensi, Ae. aegypti, Cx. quinquefasciatus, An. subpictus, Ae. albopictus, and Cx. tritaeniorhynchus with LC50 values of 61.84, 66.24, 72.47, 82.26, 89.80, and 97.95 μg/ml, respectively. B. ovalifoliolata EO was scarcely toxic to mosquito fishes, backswimmers, and water bugs predating mosquito larvae with LC50 from 4186 to 14,783 μg/ml. Overall, these results contribute to develop effective and affordable instruments to magnify the reliability of Culicidae control programs.

  6. Gene therapy in skin: choosing the optimal viral vector.

    PubMed

    Teo, Esther H; Cross, Kevin J; Bomsztyk, Elan D; Lyden, David C; Spector, Jason A

    2009-05-01

    Skin is an ideal gene therapy target because it is readily accessible and is involved in many pathologic processes. Viruses are the most common gene vectors, however, few comparative studies exist examining their efficacy in skin. This study evaluates adenovirus serotype 5, adeno-associated virus type 2 and 5, MMLV-derived retrovirus, and human immunodeficiency virus-1 derived lentivirus for gene vector activity in human dermal fibroblasts and other skin cell lines. Human immunodeficiency virus-1-based lentiviral vector resulted in over 90% transduction in all cell lines tested. Transduced cells maintained reporter expression over several passages after a single exposure. In contrast, gene activity fell rapidly over cell divisions with adenoviral and adeno-associated vectors. Therefore, lentiviral vectors are the delivery mechanism of choice for long-term therapeutic gene expression in dermal fibroblasts and other skin cell lines, whereas adenoviral or adeno-associated vectors may be preferred for short-term therapy.

  7. Vector and Axial Vector Pion Form Factors

    NASA Astrophysics Data System (ADS)

    Vitz, Michael; PEN Collaboration

    2015-04-01

    Radiative pion decay π+ -->e+ νγ (RPD) provides critical input to chiral perturbation theory (χPT). Aside from the uninteresting ``inner bremsstrahlung'' contribution from QED, the RPD rate contains ``structure dependent'' terms given by FV and FA, the vector and axial-vector pion form factors, respectively. The two appear in the decay rate in combinations FV -FA and FV +FA , i.e., in the so-called SD- and SD+ terms, respectively. The latter has been measured to high precision by the PIBETA collaboration. We report on the analysis of new data, measured by the PEN collaboration in runs between 2008 and 2010 at the Paul Scherrer Institute, Switzerland. We particularly focus on the possibility of improvement in the determination of the SD- term. Precise determinations of FV and FA test the validity of the CVC hypothesis, provide numerical input for the l9 +l10 terms in the χPT lagrangian, and constrain potential non-(V - A) terms, such as a possible tensor term FT. NSF grants PHY-0970013, 1307328, and others.

  8. Bunyavirus-vector interactions.

    PubMed

    Beaty, B J; Bishop, D H

    1988-06-01

    Recent advances in the genetics and molecular biology of bunyaviruses have been applied to understanding bunyavirus-vector interactions. Such approaches have revealed which virus gene and gene products are important in establishing infections in vectors and in transmission of viruses. However, much more information is required to understand the molecular mechanisms of persistent infections of vectors which are lifelong but apparently exert no untoward effect. In fact, it seems remarkable that LAC viral antigen can be detected in almost every cell in an ovarian follicle, yet no untoward effect on fecundity and no teratology is seen. Similarly the lifelong infection of the vector would seem to provide ample opportunity for bunyavirus evolution by genetic drift and, under the appropriate circumstances, by segment reassortment. The potential for bunyavirus evolution by segment reassortment in vectors certainly exists. For example the Group C viruses in a small forest in Brazil seem to constitute a gene pool, with the 6 viruses related alternately by HI/NT and CF reactions, which assay respectively M RNA and S RNA gene products (Casals and Whitman, 1960; Shope and Causey, 1962). Direct evidence for naturally occurring reassortant bunyaviruses has also been obtained. Oligonucleotide fingerprint analyses of field isolates of LAC virus and members of the Patois serogroup of bunyaviruses have demonstrated that reassortment does occur in nature (El Said et al., 1979; Klimas et al., 1981; Ushijima et al., 1981). Determination of the genotypic frequencies of viruses selected by the biological interactions of viruses and vectors after dual infection and segment reassortment is an important issue. Should a virus result that efficiently interacts with alternate vector species, the virus could be expressed in different circumstances with serious epidemiologic consequences. Dual infection of vectors with different viruses is not unlikely, because many bunyaviruses are sympatric in

  9. Vector meson electroproduction in QCD

    NASA Astrophysics Data System (ADS)

    Lu, Juan; Cai, Xian-Hao; Zhou, Li-Juan

    2012-08-01

    Based on the generalized QCD vector meson dominance model, we study the electroproduction of a vector meson off a proton in the QCD inspired eikonalized model. Numerical calculations for the total cross section σtot and differential cross section dσ/dt are performed for ρ, ω and varphi meson electroproduction in this paper. Since gluons interact among themselves (self-interaction), two gluons can form a glueball with quantum numbers IG, JPC = 0+,2++, decay width Γt ≈ 100 MeV, and mass of mG = 2.23 GeV. The three gluons can form a three-gluon colorless bound state with charge conjugation quantum number C = -1, called the Odderon. The mediators of interactions between projectiles (the quark and antiquark pair fluctuated from the virtual photon) and the proton target (a three-quark system) are the tensor glueball and the Odderon. Our calculated results in the tensor glueball and Odderon exchange model fit to the existing data successfully, which evidently shows that our present QCD mechanism is a good description of meson electroproduction off a proton. It should be emphasized that our mechanism is different from the theoretical framework of Block et al. We also believe that the present study and its success are important for the investigation of other vector meson electro- and photoproduction at high energies, as well as for searching for new particles such as tensor glueballs and Odderons, which have been predicted by QCD and the color glass condensate model (CGC). Therefore, in return, it can test the validity of QCD and the CGC model.

  10. Scalable motion vector coding

    NASA Astrophysics Data System (ADS)

    Barbarien, Joeri; Munteanu, Adrian; Verdicchio, Fabio; Andreopoulos, Yiannis; Cornelis, Jan P.; Schelkens, Peter

    2004-11-01

    Modern video coding applications require transmission of video data over variable-bandwidth channels to a variety of terminals with different screen resolutions and available computational power. Scalable video coding is needed to optimally support these applications. Recently proposed wavelet-based video codecs employing spatial domain motion compensated temporal filtering (SDMCTF) provide quality, resolution and frame-rate scalability while delivering compression performance comparable to that of the state-of-the-art non-scalable H.264-codec. These codecs require scalable coding of the motion vectors in order to support a large range of bit-rates with optimal compression efficiency. Scalable motion vector coding algorithms based on the integer wavelet transform followed by embedded coding of the wavelet coefficients were recently proposed. In this paper, a new and fundamentally different scalable motion vector codec (MVC) using median-based motion vector prediction is proposed. Extensive experimental results demonstrate that the proposed MVC systematically outperforms the wavelet-based state-of-the-art solutions. To be able to take advantage of the proposed scalable MVC, a rate allocation mechanism capable of optimally dividing the available rate among texture and motion information is required. Two rate allocation strategies are proposed and compared. The proposed MVC and rate allocation schemes are incorporated into an SDMCTF-based video codec and the benefits of scalable motion vector coding are experimentally demonstrated.

  11. In Vivo Cancer Targeting and Imaging-Guided Surgery with Near Infrared-Emitting Quantum Dot Bioconjugates

    PubMed Central

    Li, Yan; Li, Zhe; Wang, Xiaohui; Liu, Fengjun; Cheng, Yingsheng; Zhang, Bingbo; Shi, Donglu

    2012-01-01

    Early detection and subsequent complete surgical resection are among the most efficient methods for treating cancer. However, low detection sensitivity and incomplete tumor resection are two challenging issues. Nanoparticle-based imaging-guided surgery has proven promising for cancer-targeted imaging and subsequent debulking surgery. Particularly, the use of near infrared (NIR) fluorescent probes such as NIR quantum dots (QDs) allows deep penetration and high sensitivity for tumor detection. In this study, NIR-emitting CdTe QDs (maximum fluorescence emission peak at 728 nm) were synthesized with a high quantum yield (QY) of 38%. The tumor-specific QD bioconjugates were obtained by attaching cyclic Arg-Gly-Asp peptide (cRGD) to the surface of synthesized QDs, and then injected into U87 MG tumor-bearing mice via tail veins for tumor-targeted imaging. The tumor and its margins were visualized and distinguished by NIR QD bioconjugates, and tumor resection was successfully accomplished via NIR guidance using a Fluobeam-700 NIR imaging system. Our work indicates that the synthesized tumor-specific NIR QDs hold great promise as a potential fluorescent indicator for intraoperative tumor imaging. PMID:22916076

  12. Targeted drug delivery of Sunitinib Malate to tumor blood vessels by cRGD-chiotosan-gold nanoparticles.

    PubMed

    Saber, Mohaddeseh Mahmoudi; Bahrainian, Sara; Dinarvand, Rassoul; Atyabi, Fatemeh

    2017-01-30

    The unique characteristics of tumor vasculature represent an attractive strategy for targeted delivery of antitumor and antiangiogenic agents to the tumor. The purpose of this study was to prepare c(RGDfK) labeled chitosan capped gold nanoparticles [cRGD(CS-Au) NPs] as a carrier for selective intracellular delivery of Sunitinib Malate (STB) to the tumor vasculature. cRGD(CS-Au) NPs was formed by electrostatic interaction between cationic CS and anionic AuNPs. cRGD modified CS-Au NPs had a spherical shape with a narrow size distribution. The entrapment efficiency of sunitinib molecule was found to be 45.2%±2.05. Confocal microscopy showed enhanced and selective uptake of cRGD(CS-Au) NPs into MCF-7 and HUVEC cells compared with non-targeted CS-Au NPs. Our results suggest that it may be possible to use cRGD(CS-Au) NPs as a carrier for delivery of anticancer drugs, genes and biomolecules for inhibiting tumor vasculature. Copyright © 2016. Published by Elsevier B.V.

  13. Feature clustering in direct eigen-vector data reduction using support vector machines

    NASA Astrophysics Data System (ADS)

    Riasati, Vahid R.; Gao, Wenhue

    2012-06-01

    Principal Component Analysis (PCA) has been used in a variety of applications like feature extraction for classification, data compression and dimensionality reduction. Often, a small set of principal components are sufficient to capture the largest variations in the data. As a result, the eigen-values of the data covariance matrix with the lowest magnitude are ignored (along with their corresponding eigen-vectors) and the remaining eigenvectors are used for a 'coarse' representation of the data. It is well known that this process of choosing a few principal components naturally induces a loss in information from a signal reconstruction standpoint. We propose a new technique to represent the data in terms of a new set of basis vectors where the high-frequency detail is preserved, at the expense of a 'feature-scale blurring'. In other words, the 'blurring' that occurs due to possible colinearities in the bases vectors is relative to the eigen-features' scales; this is inherently different from a systematic blurring function. Instead of thresholding the eigen-values, we retain all eigen-values, and apply thresholds on the components of each eigen-vector separately. The resulting basis vectors can no longer be interpreted as eigenvectors and will, in general, lose their orthogonality properties, but offer benefits in terms of preserving detail that is crucial for classification tasks. We test the merits of this new basis representation for magnitude synthetic aperture radar (SAR) Automatic Target Recognition (ATR). A feature vector is obtained by projecting a SAR image onto the aforementioned basis. Decision engines such as support vector machines (SVMs) are trained on example feature vectors per class and ultimately used to recognize the target class in real-time. Experimental validation are performed on the MSTAR database and involve comparisons against a PCA based ATR algorithm.

  14. Replicon RNA Viral Vectors as Vaccines

    PubMed Central

    Lundstrom, Kenneth

    2016-01-01

    Single-stranded RNA viruses of both positive and negative polarity have been used as vectors for vaccine development. In this context, alphaviruses, flaviviruses, measles virus and rhabdoviruses have been engineered for expression of surface protein genes and antigens. Administration of replicon RNA vectors has resulted in strong immune responses and generation of neutralizing antibodies in various animal models. Immunization of mice, chicken, pigs and primates with virus-like particles, naked RNA or layered DNA/RNA plasmids has provided protection against challenges with lethal doses of infectious agents and administered tumor cells. Both prophylactic and therapeutic efficacy has been achieved in cancer immunotherapy. Moreover, recombinant particles and replicon RNAs have been encapsulated by liposomes to improve delivery and targeting. Replicon RNA vectors have also been subjected to clinical trials. Overall, immunization with self-replicating RNA viruses provides high transient expression levels of antigens resulting in generation of neutralizing antibody responses and protection against lethal challenges under safe conditions. PMID:27827980

  15. Lentiviral vectors that carry anti-HIV shRNAs: problems and solutions.

    PubMed

    ter Brake, Olivier; Berkhout, Ben

    2007-09-01

    HIV-1 replication can be inhibited with RNA interference (RNAi) by expression of short hairpin RNA (shRNA) from a lentiviral vector. Because lentiviral vectors are based on HIV-1, viral sequences in the vector system are potential targets for the antiviral shRNAs. Here, we investigated all possible routes by which shRNAs can target the lentiviral vector system. Expression cassettes for validated shRNAs with targets within HIV-1 Leader, Gag-Pol, Tat/Rev and Nef sequences were inserted in the lentiviral vector genome. Third-generation self-inactivating HIV-1-based lentiviral vectors were produced and lentiviral vector capsid production and transduction titer determined. RNAi against HIV-1 sequences within the vector backbone results in a reduced transduction titer while capsid production was unaffected. The notable exception is self-targeting of the shRNA encoding sequence, which does not affect transduction titer. This is due to folding of the stable shRNA hairpin structure, which masks the target for the RNAi machinery. Targeting of Gag-Pol mRNA reduces both capsid production and transduction titer, which was improved with a human codon-optimized Gag-Pol construct. When Rev mRNA was targeted, no reduction in capsid production and transduction titer was observed. Lentiviral vector titers can be negatively affected when shRNAs against the vector backbone and the Gag-Pol mRNA are expressed during lentiviral vector production. Titer reductions due to targeting of the Gag-Pol mRNA can be avoided with a human codon-optimized Gag-Pol packaging plasmid. The remaining targets in the vector backbone may be modified by point mutations to resist RNAi-mediated degradation during vector production.

  16. Eco-friendly drugs from the marine environment: spongeweed-synthesized silver nanoparticles are highly effective on Plasmodium falciparum and its vector Anopheles stephensi, with little non-target effects on predatory copepods.

    PubMed

    Murugan, Kadarkarai; Panneerselvam, Chellasamy; Subramaniam, Jayapal; Madhiyazhagan, Pari; Hwang, Jiang-Shiou; Wang, Lan; Dinesh, Devakumar; Suresh, Udaiyan; Roni, Mathath; Higuchi, Akon; Nicoletti, Marcello; Benelli, Giovanni

    2016-08-01

    Mosquitoes act as vectors of devastating pathogens and parasites, representing a key threat for millions of humans and animals worldwide. The control of mosquito-borne diseases is facing a number of crucial challenges, including the emergence of artemisinin and chloroquine resistance in Plasmodium parasites, as well as the presence of mosquito vectors resistant to synthetic and microbial pesticides. Therefore, eco-friendly tools are urgently required. Here, a synergic approach relying to nanotechnologies and biological control strategies is proposed. The marine environment is an outstanding reservoir of bioactive natural products, which have many applications against pests, parasites, and pathogens. We proposed a novel method of seaweed-mediated synthesis of silver nanoparticles (AgNP) using the spongeweed Codium tomentosum, acting as a reducing and capping agent. AgNP were characterized by UV-Vis spectroscopy, Fourier transform infrared (FTIR) spectroscopy, scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDX), and X-ray diffraction (XRD). In mosquitocidal assays, the 50 % lethal concentration (LC50) of C. tomentosum extract against Anopheles stephensi ranged from 255.1 (larva I) to 487.1 ppm (pupa). LC50 of C. tomentosum-synthesized AgNP ranged from 18.1 (larva I) to 40.7 ppm (pupa). In laboratory, the predation efficiency of Mesocyclops aspericornis copepods against A. stephensi larvae was 81, 65, 17, and 9 % (I, II, III, and IV instar, respectively). In AgNP contaminated environment, predation was not affected; 83, 66, 19, and 11 % (I, II, III, and IV). The anti-plasmodial activity of C. tomentosum extract and spongeweed-synthesized AgNP was evaluated against CQ-resistant (CQ-r) and CQ-sensitive (CQ-s) strains of Plasmodium falciparum. Fifty percent inhibitory concentration (IC50) of C. tomentosum were 51.34 μg/ml (CQ-s) and 65.17 μg/ml (CQ-r); C. tomentosum-synthesized AgNP achieved IC50 of 72.45 μg/ml (CQ-s) and 76.08

  17. Vector financial rogue waves

    NASA Astrophysics Data System (ADS)

    Yan, Zhenya

    2011-11-01

    The coupled nonlinear volatility and option pricing model presented recently by Ivancevic is investigated, which generates a leverage effect, i.e., stock volatility is (negatively) correlated to stock returns, and can be regarded as a coupled nonlinear wave alternative of the Black-Scholes option pricing model. In this Letter, we analytically propose vector financial rogue waves of the coupled nonlinear volatility and option pricing model without an embedded w-learning. Moreover, we exhibit their dynamical behaviors for chosen different parameters. The vector financial rogue wave (rogon) solutions may be used to describe the possible physical mechanisms for the rogue wave phenomena and to further excite the possibility of relative researches and potential applications of vector rogue waves in the financial markets and other related fields.

  18. Vectors--shuttle vehicles for gene therapy.

    PubMed

    Wilson, J M

    1997-01-01

    Gene therapy is being considered for the treatment of various inherited and acquired disorders. The basic premise of this new therapeutic modality is manipulation of gene expression towards a therapeutic end. The early development of the field focused on a technique called ex vivo gene therapy in which autologous cells are genetically manipulated in culture prior to transplantation. Recent advances have stimulated the development of in vivo gene therapy approaches based on direct delivery of the therapeutic gene to cells in vivo. The rate-limiting technologies of gene therapy are the gene delivery vehicles, called vectors, used to accomplish gene transfer. The most efficient vectors are based on recombinant versions of viruses with retroviral vectors serving as prototypes. This viral vector system has been exploited in ex vivo approaches of gene therapy in which cultured, dividing cells are transduced with the recombinant virus resulting in integration of the proviral DNA into the chromosomal DNA of the recipient cell. The use of retroviral vectors in gene therapy has been restricted to ex vivo approaches because of difficulties in purifying the virion and the requirement that the target cell is dividing at the time of transduction. More recently, vectors based on adenoviruses have been developed for in vivo gene therapy. These viruses can be grown in large quantities and highly purified. Importantly, they efficiently transduce the recombinant genome into non-dividing cells. Applications include in vivo gene delivery to a variety of targets such as muscle, lung, liver and the central nervous system. Clinical trials of in vivo delivery with adenoviruses have been undertaken for the treatment of cystic fibrosis.

  19. Enhancing titres of therapeutic viral vectors using the transgene repression in vector production (TRiP) system

    PubMed Central

    Maunder, H. E.; Wright, J.; Kolli, B. R.; Vieira, C. R.; Mkandawire, T. T.; Tatoris, S.; Kennedy, V.; Iqball, S.; Devarajan, G.; Ellis, S.; Lad, Y.; Clarkson, N. G.; Mitrophanous, K. A.; Farley, D. C.

    2017-01-01

    A key challenge in the field of therapeutic viral vector/vaccine manufacturing is maximizing production. For most vector platforms, the ‘benchmark' vector titres are achieved with inert reporter genes. However, expression of therapeutic transgenes can often adversely affect vector titres due to biological effects on cell metabolism and/or on the vector virion itself. Here, we exemplify the novel ‘Transgene Repression In vector Production' (TRiP) system for the production of both RNA- and DNA-based viral vectors. The TRiP system utilizes a translational block of one or more transgenes by employing the bacterial tryptophan RNA-binding attenuation protein (TRAP), which binds its target RNA sequence close to the transgene initiation codon. We report enhancement of titres of lentiviral vectors expressing Cyclo-oxygenase-2 by 600-fold, and adenoviral vectors expressing the pro-apoptotic gene Bax by >150,000-fold. The TRiP system is transgene-independent and will be a particularly useful platform in the clinical development of viral vectors expressing problematic transgenes. PMID:28345582

  20. Purification of recombinant adeno-associated virus type 8 vectors by ion exchange chromatography generates clinical grade vector stock.

    PubMed

    Davidoff, Andrew M; Ng, Catherine Y C; Sleep, Susan; Gray, John; Azam, Selina; Zhao, Yuan; McIntosh, Jenny H; Karimipoor, Morteza; Nathwani, Amit C

    2004-11-01

    Recombinant vectors based on the recently isolated AAV serotype 8 (rAAV-8) shows great promise for gene therapy, particularly for disorders affecting the liver. Transition of this vector system to the clinic, however, is limited by the lack of an efficient scaleable purification method. In this report, we describe a simple method for purification of rAAV-8 vector particles based on ion exchange chromatography that generates vector stocks with greater than 90% purity. The average yield of purified rAAV-8 from five different vector preparation was 41%. Electron microscopy of these purified stocks revealed typical icosohedral virions with less than 10% empty particles. Liver targeted delivery of ion-exchange purified rAAV-8 vector encoding the human factor IX (hFIX) gene, resulted in plasma hFIX levels approaching 30% of normal in immunocompetent mice, which is 20-fold higher than observed with an equivalent number of rAAV-5 ion exchange purified vector particles. The method takes less then 5 h to process and purify rAAV-8 vector from producer cells and represents a significant advance on the CsCl density centrifugation technique in current use for purification of rAAV-8 vector systems and will likely facilitate the transition of the rAAV-8 vector system to the clinic.

  1. Scalar-vector bootstrap

    NASA Astrophysics Data System (ADS)

    Rejon-Barrera, Fernando; Robbins, Daniel

    2016-01-01

    We work out all of the details required for implementation of the conformal bootstrap program applied to the four-point function of two scalars and two vectors in an abstract conformal field theory in arbitrary dimension. This includes a review of which tensor structures make appearances, a construction of the projectors onto the required mixed symmetry representations, and a computation of the conformal blocks for all possible operators which can be exchanged. These blocks are presented as differential operators acting upon the previously known scalar conformal blocks. Finally, we set up the bootstrap equations which implement crossing symmetry. Special attention is given to the case of conserved vectors, where several simplifications occur.

  2. Bunyavirus-Vector Interactions

    PubMed Central

    Horne, Kate McElroy; Vanlandingham, Dana L.

    2014-01-01

    The Bunyaviridae family is comprised of more than 350 viruses, of which many within the Hantavirus, Orthobunyavirus, Nairovirus, Tospovirus, and Phlebovirus genera are significant human or agricultural pathogens. The viruses within the Orthobunyavirus, Nairovirus, and Phlebovirus genera are transmitted by hematophagous arthropods, such as mosquitoes, midges, flies, and ticks, and their associated arthropods not only serve as vectors but also as virus reservoirs in many cases. This review presents an overview of several important emerging or re-emerging bunyaviruses and describes what is known about bunyavirus-vector interactions based on epidemiological, ultrastructural, and genetic studies of members of this virus family. PMID:25402172

  3. cRGD-installed docetaxel-loaded mertansine prodrug micelles: redox-triggered ratiometric dual drug release and targeted synergistic treatment of B16F10 melanoma

    NASA Astrophysics Data System (ADS)

    Zhong, Ping; Qiu, Min; Zhang, Jian; Sun, Huanli; Cheng, Ru; Deng, Chao; Meng, Fenghua; Zhong, Zhiyuan

    2017-07-01

    Combinatorial chemotherapy, which has emerged as a promising treatment modality for intractable cancers, is challenged by a lack of tumor-targeting, robust and ratiometric dual drug release systems. Here, docetaxel-loaded cRGD peptide-decorated redox-activable micellar mertansine prodrug (DTX-cRGD-MMP) was developed for targeted and synergistic treatment of B16F10 melanoma-bearing C57BL/6 mice. DTX-cRGD-MMP exhibited a small size of ca. 49 nm, high DTX and DM1 loading, low drug leakage under physiological conditions, with rapid release of both DTX and DM1 under a cytoplasmic reductive environment. Notably, MTT and flow cytometry assays showed that DTX-cRGD-MMP brought about a synergistic antitumor effect to B16F10 cancer cells, with a combination index of 0.37 and an IC50 over 3- and 13-fold lower than cRGD-MMP (w/o DTX) and DTX-cRGD-Ms (w/o DM1) controls, respectively. In vivo studies revealed that DTX-cRGD-MMP had a long circulation time and a markedly improved accumulation in the B16F10 tumor compared with the non-targeting DTX-MMP control (9.15 versus 3.13% ID/g at 12 h post-injection). Interestingly, mice treated with DTX-cRGD-MMP showed almost complete growth inhibition of B16F10 melanoma, with tumor inhibition efficacy following an order of DTX-cRGD-MMP > DTX-MMP (w/o cRGD) > cRGD-MMP (w/o DTX) > DTX-cRGD-Ms (w/o DM1) > free DTX. Consequently, DTX-cRGD-MMP significantly improved the survival rates of B16F10 melanoma-bearing mice. Importantly, DTX-cRGD-MMP caused little adverse effects as revealed by mice body weights and histological analyses. The combination of two mitotic inhibitors, DTX and DM1, appears to be an interesting approach for effective cancer therapy.

  4. Viral Vectors for in Vivo Gene Transfer

    NASA Astrophysics Data System (ADS)

    Thévenot, E.; Dufour, N.; Déglon, N.

    The transfer of DNA into the nucleus of a eukaryotic cell (gene transfer) is a central theme of modern biology. The transfer is said to be somatic when it refers to non-germline organs of a developed individual, and germline when it concerns gametes or the fertilised egg of an animal, with the aim of transmitting the relevant genetic modification to its descendents [1]. The efficient introduction of genetic material into a somatic or germline cell and the control of its expression over time have led to major advances in understanding how genes work in vivo, i.e., in living organisms (functional genomics), but also to the development of innovative therapeutic methods (gene therapy). The efficiency of gene transfer is conditioned by the vehicle used, called the vector. Desirable features for a vector are as follows: Easy to produce high titer stocks of the vector in a reproducible way. Absence of toxicity related to transduction (transfer of genetic material into the target cell, and its expression there) and no immune reaction of the organism against the vector and/or therapeutic protein. Stability in the expression of the relevant gene over time, and the possibility of regulation, e.g., to control expression of the therapeutic protein on the physiological level, or to end expression at the end of treatment. Transduction of quiescent cells should be as efficient as transduction of dividing cells. Vectors currently used fall into two categories: non-viral and viral vectors. In non-viral vectors, the DNA is complexed with polymers, lipids, or cationic detergents (described in Chap. 3). These vectors have a low risk of toxicity and immune reaction. However, they are less efficient in vivo than viral vectors when it comes to the number of cells transduced and long-term transgene expression. (Naked DNA transfer or electroporation is rather inefficient in the organism. This type of gene transfer will not be discussed here, and the interested reader is referred to the

  5. Malaria vector species in Colombia - A review

    PubMed Central

    Montoya-Lerma, James; Solarte, Yezid A; Giraldo-Calderón, Gloria Isabel; Quiñones, Martha L; Ruiz-López, Freddy; Wilkerson, Richard C; González, Ranulfo

    2016-01-01

    Here we present a comprehensive review of the literature on the vectorial importance of the major Anopheles malaria vectors in Colombia. We provide basic information on the geographical distribution, altitudinal range, immature habitats, adult behaviour, feeding preferences and anthropophily, endophily and infectivity rates. We additionally review information on the life cycle, longevity and population fluctuation of Colombian Anopheles species. Emphasis was placed on the primary vectors that have been epidemiologically incriminated in malaria transmission: Anopheles darlingi, Anopheles albimanus and Anopheles nuneztovari. The role of a selection of local, regional or secondary vectors (e.g., Anopheles pseudopunctipennis and Anopheles neivai) is also discussed. We highlight the importance of combining biological, morphological and molecular data for the correct taxonomical determination of a given species, particularly for members of the species complexes. We likewise emphasise the importance of studying the bionomics of primary and secondary vectors along with an examination of the local conditions affecting the transmission of malaria. The presence and spread of the major vectors and the emergence of secondary species capable of transmitting human Plasmodia are of great interest. When selecting control measures, the anopheline diversity in the region must be considered. Variation in macroclimate conditions over a species’ geographical range must be well understood and targeted to plan effective control measures based on the population dynamics of the local Anopheles species. PMID:21881778

  6. [Lipid vectors. New strategies for gene therapy].

    PubMed

    Romero, E L; Morilla, M J; Bakas, L S

    2001-01-01

    Phospholipids are capable of spontaneous self-assembling, a remarkable differential property if compared with the rest of biological molecules. By their means it is relatively easy to generate extremely stable sealed structures, with controlled shape, size and packing, known as liposomes. In this article, we review the use of liposomes to improve the transfection process in eucaryotic cells, in vitro as well as in vivo. By employing lipid vectors, it is feasible to selectively transport a DNA segment to any target of the body, to force it to enter a cell and once inside it, to exert a control on its ultimate intracellular fate. The goal of lipid vectors to successfully transfect a cell in vivo, lies on the provision of a mechanical protection for DNA against plasma degradation, together with the possibility of controlling DNA biodistribution, independently of its size and sequence. Moreover, lipid vectors are not carcinogenic and are poorly immunogenic. Current challenge in lipid synthesis allows for a vector design which should be efficient enough to compete with high transfection levels of a viral vector, but with the extreme versatility, simplicity and biosafety characteristic of self assembling molecules.

  7. Support vector machines

    NASA Technical Reports Server (NTRS)

    Garay, Michael J.; Mazzoni, Dominic; Davies, Roger; Wagstaff, Kiri

    2004-01-01

    Support Vector Machines (SVMs) are a type of supervised learning algorith,, other examples of which are Artificial Neural Networks (ANNs), Decision Trees, and Naive Bayesian Classifiers. Supervised learning algorithms are used to classify objects labled by a 'supervisor' - typically a human 'expert.'.

  8. Vectors Point Toward Pisa

    ERIC Educational Resources Information Center

    Dean, Richard A.

    1971-01-01

    The author shows that the set of all sequences in which each term is the sum of the two previous terms forms a vector space of dimension two. He uses this result to obtain the formula for the Fibonacci sequence and applies the same technique to other linear recursive relations. (MM)

  9. Killing vectors and anisotropy

    SciTech Connect

    Krisch, J. P.; Glass, E. N.

    2009-08-15

    We consider an action that can generate fluids with three unequal stresses for metrics with a spacelike Killing vector. The parameters in the action are directly related to the stress anisotropies. The field equations following from the action are applied to an anisotropic cosmological expansion and an extension of the Gott-Hiscock cosmic string.

  10. Production of lentiviral vectors

    PubMed Central

    Merten, Otto-Wilhelm; Hebben, Matthias; Bovolenta, Chiara

    2016-01-01

    Lentiviral vectors (LV) have seen considerably increase in use as gene therapy vectors for the treatment of acquired and inherited diseases. This review presents the state of the art of the production of these vectors with particular emphasis on their large-scale production for clinical purposes. In contrast to oncoretroviral vectors, which are produced using stable producer cell lines, clinical-grade LV are in most of the cases produced by transient transfection of 293 or 293T cells grown in cell factories. However, more recent developments, also, tend to use hollow fiber reactor, suspension culture processes, and the implementation of stable producer cell lines. As is customary for the biotech industry, rather sophisticated downstream processing protocols have been established to remove any undesirable process-derived contaminant, such as plasmid or host cell DNA or host cell proteins. This review compares published large-scale production and purification processes of LV and presents their process performances. Furthermore, developments in the domain of stable cell lines and their way to the use of production vehicles of clinical material will be presented. PMID:27110581

  11. The vector reflector.

    PubMed

    Citrin, D S

    2012-06-15

    A linearly polarized Bessel beam, whose spatial frequencies correspond to the Brewster angle, impinging at normal incidence on a higher refractive-index interface is shown to lead to a reflected field that can be used to produce an azimuthally polarized optical vector beam.

  12. LTR-vectors

    SciTech Connect

    Vande Woude, G.F.; McClements, W.L.; Oskarsson, M.K.; Blair, D.G.

    1981-07-01

    The patent application describes the production of vectors composed of portions of retrovirus, particularly of Moloney sarcoma virus DNA including the 'LTR' sequence which can activate genes and additional viral sequences which can 'rescue' these genes into a replicating virus particle.

  13. Support vector machines

    NASA Technical Reports Server (NTRS)

    Garay, Michael J.; Mazzoni, Dominic; Davies, Roger; Wagstaff, Kiri

    2004-01-01

    Support Vector Machines (SVMs) are a type of supervised learning algorith,, other examples of which are Artificial Neural Networks (ANNs), Decision Trees, and Naive Bayesian Classifiers. Supervised learning algorithms are used to classify objects labled by a 'supervisor' - typically a human 'expert.'.

  14. Singular Vectors' Subtle Secrets

    ERIC Educational Resources Information Center

    James, David; Lachance, Michael; Remski, Joan

    2011-01-01

    Social scientists use adjacency tables to discover influence networks within and among groups. Building on work by Moler and Morrison, we use ordered pairs from the components of the first and second singular vectors of adjacency matrices as tools to distinguish these groups and to identify particularly strong or weak individuals.

  15. What is a vector?

    PubMed

    Wilson, Anthony James; Morgan, Eric René; Booth, Mark; Norman, Rachel; Perkins, Sarah Elizabeth; Hauffe, Heidi Christine; Mideo, Nicole; Antonovics, Janis; McCallum, Hamish; Fenton, Andy

    2017-05-05

    Many important and rapidly emerging pathogens of humans, livestock and wildlife are 'vector-borne'. However, the term 'vector' has been applied to diverse agents in a broad range of epidemiological systems. In this perspective, we briefly review some common definitions, identify the strengths and weaknesses of each and consider the functional differences between vectors and other hosts from a range of ecological, evolutionary and public health perspectives. We then consider how the use of designations can afford insights into our understanding of epidemiological and evolutionary processes that are not otherwise apparent. We conclude that from a medical and veterinary perspective, a combination of the 'haematophagous arthropod' and 'mobility' definitions is most useful because it offers important insights into contact structure and control and emphasizes the opportunities for pathogen shifts among taxonomically similar species with similar feeding modes and internal environments. From a population dynamics and evolutionary perspective, we suggest that a combination of the 'micropredator' and 'sequential' definition is most appropriate because it captures the key aspects of transmission biology and fitness consequences for the pathogen and vector itself. However, we explicitly recognize that the value of a definition always depends on the research question under study.This article is part of the themed issue 'Opening the black box: re-examining the ecology and evolution of parasite transmission'. © 2017 The Authors.

  16. Vector-borne diseases.

    PubMed

    Gubler, D J

    2009-08-01

    Vector-borne diseases have been the scourge of man and animals since the beginning of time. Historically, these are the diseases that caused the great plagues such as the 'Black Death' in Europe in the 14th Century and the epidemics of yellow fever that plagued the development of the New World. Others, such as Nagana, contributed to the lack of development in Africa for many years. At the turn of the 20th Century, vector-borne diseases were among the most serious public and animal health problems in the world. For the most part, these diseases were controlled by the middle of the 20th Century through the application of knowledge about their natural history along with the judicious use of DDT (dichlorodiphenyltrichloroethane) and other residual insecticides to interrupt the transmission cycle between arthropod and vertebrate host. However, this success initiated a period of complacency in the 1960s and 1970s, which resulted in the redirection of resources away from prevention and control of vector-borne diseases. The 1970s was also a time in which there were major changes to public health policy. Global trends, combined with changes in animal husbandry, urbanisation, modern transportation and globalisation, have resulted in a global re-emergence of epidemic vector-borne diseases affecting both humans and animals over the past 30 years.

  17. Singular Vectors' Subtle Secrets

    ERIC Educational Resources Information Center

    James, David; Lachance, Michael; Remski, Joan

    2011-01-01

    Social scientists use adjacency tables to discover influence networks within and among groups. Building on work by Moler and Morrison, we use ordered pairs from the components of the first and second singular vectors of adjacency matrices as tools to distinguish these groups and to identify particularly strong or weak individuals.

  18. Vector potential methods

    NASA Technical Reports Server (NTRS)

    Hafez, M.

    1989-01-01

    Vector potential and related methods, for the simulation of both inviscid and viscous flows over aerodynamic configurations, are briefly reviewed. The advantages and disadvantages of several formulations are discussed and alternate strategies are recommended. Scalar potential, modified potential, alternate formulations of Euler equations, least-squares formulation, variational principles, iterative techniques and related methods, and viscous flow simulation are discussed.

  19. AAV Vectorization of DSB-mediated Gene Editing Technologies.

    PubMed

    Moser, Rachel J; Hirsch, Matthew L

    2016-01-01

    Recent work both at the bench and the bedside demonstrate zinc-finger nucleases (ZFNs), CRISPR/Cas9, and other programmable site-specific endonuclease technologies are being successfully utilized within and alongside AAV vectors to induce therapeutically relevant levels of directed gene editing within the human chromosome. Studies from past decades acknowledge that AAV vector genomes are enhanced substrates for homology-directed repair in the presence or absence of targeted DNA damage within the host genome. Additionally, AAV vectors are currently the most efficient format for in vivo gene delivery with no vector related complications in >100 clinical trials for diverse diseases. At the same time, advancements in the design of custom-engineered site-specific endonucleases and the utilization of elucidated endonuclease formats have resulted in efficient and facile genetic engineering for basic science and for clinical therapies. AAV vectors and gene editing technologies are an obvious marriage, using AAV for the delivery of repair substrate and/or a gene encoding a designer endonuclease; however, while efficient delivery and enhanced gene targeting by vector genomes are advantageous, other attributes of AAV vectors are less desirable for gene editing technologies. This review summarizes the various roles that AAV vectors play in gene editing technologies and provides insight into its trending applications for the treatment of genetic diseases.

  20. Evaluation of Attractive Toxic Sugar Bait (ATSB) - Barrier for Control of Vector and Nuisance Mosquitoes and Its Effect on Non-target Organisms in Sub-tropical Environments in Florida

    DTIC Science & Technology

    2014-01-01

    target organisms in sub-tropical environments in Florida Whitney A. Quallsa,∗, Günter C. Müllerb, Edita E. Revayc, Sandra A. Alland...sub-tropical environments in Florida 5a. CONTRACT NUMBER 5b. GRANT NUMBER 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) 5d. PROJECT NUMBER 5e. TASK...control of anopheline mosquitoes using ATSB has not yet been demonstrated in more tropical, sub-tropical environments like Latin America or Florida , US

  1. Characterization of tumor-targeting Ag2S quantum dots for cancer imaging and therapy in vivo

    NASA Astrophysics Data System (ADS)

    Chen, Haiyan; Li, Bowen; Zhang, Min; Sun, Kang; Wang, Yiran; Peng, Kerui; Ao, Mengdi; Guo, Yiran; Gu, Yueqing

    2014-10-01

    Nanomedicine platforms that have the potential to simultaneously provide the function of molecular imaging and therapeutic treatment in one system are beneficial to address the challenges of cancer heterogeneity and adaptive resistance. In this study, Cyclic RGD peptide (cRGD), a less-expensive active tumor targeting tri-peptide, and doxorubicin (DOX), a widely used chemotherapeutic drug, were covalently attached to Ag2S quantum dots (QDs) to form the nano-conjugates Ag2S-DOX-cRGD. The optical characterization of Ag2S-DOX-cRGD manifested the maintenance of QDs fluorescence, which suggested the potential of Ag2S for monitoring intracellular and systemic drug distribution. The low biotoxicity of Ag2S QDs indicated that they are promisingly safe nanoparticles for bio-applications. Furthermore, the selective imaging and favorable tumor inhibition of the nanoconjugates were demonstrated at both cell and animal levels. These results indicated a promising future for the utilization of Ag2S QDs as a kind of multi-functional nano platform to achieve imaging-visible nano-therapeutics.

  2. Vector fields in cosmology

    NASA Astrophysics Data System (ADS)

    Davydov, E. A.

    2012-06-01

    Vector fields can arise in the cosmological context in different ways, and we discuss both abelian and nonabelian sector. In the abelian sector vector fields of the geometrical origin (from dimensional reduction and Einstein-Eddington modification of gravity) can provide a very non-trivial dynamics, which can be expressed in terms of the effective dilaton-scalar gravity with the specific potential. In the non-abelian sector we investigate the Yang-Mills SU(2) theory which admits isotropic and homogeneous configuration. Provided the non-linear dependence of the lagrangian on the invariant FμνF~μν, one can obtain the inflationary regime with the exponential growth of the scale factor. The effective amplitudes of the `electric' and `magnetic' components behave like slowly varying scalars at this regime, what allows the consideration of some realistic models with non-linear terms in the Yang-Mills lagrangian.

  3. Vehicle Based Vector Sensor

    DTIC Science & Technology

    2015-09-28

    300001 1 of 16 VEHICLE-BASED VECTOR SENSOR STATEMENT OF GOVERNMENT INTEREST [0001] The invention described herein may be manufactured and...CROSS REFERENCE TO OTHER PATENT APPLICATIONS [0002] None. BACKGROUND OF THE INVENTION (1) Field of the Invention [0003] The invention is an... invention , is a small volume of fluid surrounding a point where averaged properties (e.g., velocity, temperature, etc.) can be analyzed with continuum

  4. Vector Magnetograph Design

    NASA Technical Reports Server (NTRS)

    Chipman, Russell A.

    1996-01-01

    This report covers work performed during the period of November 1994 through March 1996 on the design of a Space-borne Solar Vector Magnetograph. This work has been performed as part of a design team under the supervision of Dr. Mona Hagyard and Dr. Alan Gary of the Space Science Laboratory. Many tasks were performed and this report documents the results from some of those tasks, each contained in the corresponding appendix. Appendices are organized in chronological order.

  5. Some experiences with Krylov vectors and Lanczos vectors

    NASA Technical Reports Server (NTRS)

    Craig, Roy R., Jr.; Su, Tzu-Jeng; Kim, Hyoung M.

    1993-01-01

    This paper illustrates the use of Krylov vectors and Lanczos vectors for reduced-order modeling in structural dynamics and for control of flexible structures. Krylov vectors and Lanczos vectors are defined and illustrated, and several applications that have been under study at The University of Texas at Austin are reviewed: model reduction for undamped structural dynamics systems, component mode synthesis using Krylov vectors, model reduction of damped structural dynamics systems, and one-sided and two-sided unsymmetric block-Lanczos model-reduction algorithms.

  6. Viral vectors for gene transfer: current status of gene therapeutics.

    PubMed

    Heilbronn, Regine; Weger, Stefan

    2010-01-01

    Gene therapy for the correction of inherited or acquired disease has gained increasing importance in recent years. Successful treatment of children suffering from severe combined immunodeficiency (SCID) was achieved using retrovirus vectors for gene transfer. Encouraging improvements of vision were reported in a genetic eye disorder (LCA) leading to early childhood blindness. Adeno-associated virus (AAV) vectors were used for gene transfer in these trials. This chapter gives an overview of the design and delivery of viral vectors for the transport of a therapeutic gene into a target cell or tissue. The construction and production of retrovirus, lentivirus, and AAV vectors are covered. The focus is on production methods suitable for biopharmaceutical upscaling and for downstream processing. Quality control measures and biological safety considerations for the use of vectors in clinical trials are discussed.

  7. Isomap based supporting vector machine

    NASA Astrophysics Data System (ADS)

    Liang, W. N.

    2015-12-01

    This research presents a new isomap based supporting vector machine method. Isomap is a dimension reduction method which is able to analyze nonlinear relationship of data on manifolds. Accordingly, support vector machine is established on the isomap manifold to classify given and predict unknown data. A case study of the isomap based supporting vector machine for environmental planning problems is conducted.

  8. Dipole-mode vector solitons

    PubMed

    Garcia-Ripoll; Perez-Garcia; Ostrovskaya; Kivshar

    2000-07-03

    We find a new type of optical vector soliton that originates from trapping of a dipole mode by the soliton-induced waveguides. These solitons, which appear as a consequence of the vector nature of the two-component system, are more stable than the previously found optical vortex solitons and represent a new type of extremely robust nonlinear vector structure.

  9. What is a vector?

    PubMed Central

    Morgan, Eric René; Booth, Mark; Norman, Rachel; Mideo, Nicole; McCallum, Hamish; Fenton, Andy

    2017-01-01

    Many important and rapidly emerging pathogens of humans, livestock and wildlife are ‘vector-borne’. However, the term ‘vector’ has been applied to diverse agents in a broad range of epidemiological systems. In this perspective, we briefly review some common definitions, identify the strengths and weaknesses of each and consider the functional differences between vectors and other hosts from a range of ecological, evolutionary and public health perspectives. We then consider how the use of designations can afford insights into our understanding of epidemiological and evolutionary processes that are not otherwise apparent. We conclude that from a medical and veterinary perspective, a combination of the ‘haematophagous arthropod’ and ‘mobility’ definitions is most useful because it offers important insights into contact structure and control and emphasizes the opportunities for pathogen shifts among taxonomically similar species with similar feeding modes and internal environments. From a population dynamics and evolutionary perspective, we suggest that a combination of the ‘micropredator’ and ‘sequential’ definition is most appropriate because it captures the key aspects of transmission biology and fitness consequences for the pathogen and vector itself. However, we explicitly recognize that the value of a definition always depends on the research question under study. This article is part of the themed issue ‘Opening the black box: re-examining the ecology and evolution of parasite transmission’. PMID:28289253

  10. Vector Helmholtz-Gauss and vector Laplace-Gauss beams.

    PubMed

    Bandres, Miguel A; Gutiérrez-Vega, Julio C

    2005-08-15

    We demonstrate the existence of vector Helmholtz-Gauss (vHzG) and vector Laplace-Gauss beams that constitute two general families of localized vector beam solutions of the Maxwell equations in the paraxial approximation. The electromagnetic components are determined starting from the scalar solutions of the two-dimensional Helmholtz and Laplace equations, respectively. Special cases of the vHzG beams are TE and TM Gaussian vector beams, nondiffracting vector Bessel beams, polarized Bessel-Gauss beams, modes in cylindrical waveguides and cavities, and scalar Helmholtz-Gauss beams. The general expression of the vHzG beams can be used straightforwardly to obtain vector Mathieu-Gauss and vector parabolic-Gauss beams, which to our knowledge have not yet been reported.

  11. Vector meson modification in nuclear matter at CLAS

    SciTech Connect

    Djalali, Chaden; Wood, Michael; Nasseripour, Rakhsha; Weygand, Dennis

    2008-09-01

    Photoproduction of vector mesons off nuclei were performed at Jefferson Lab using the CEBAF Large Acceptance Spectrometer (CLAS). The properties of the A vector mesons were investigated via their rare leptonic decay to e+e . After subtracting the combinatorial background, the A meson mass distributions were extracted for each of the targets. We observe no effects on the mass of the A meson, some widening in titanium and iron is observed consistent with the collisional broadening.

  12. Vector representation of tourmaline compositions

    NASA Technical Reports Server (NTRS)

    Burt, Donald M.

    1989-01-01

    The vector method for representing mineral compositions of amphibole and mica groups is applied to the tourmaline group. Consideration is given to the methods for drawing the relevant vector diagrams, relating the exchange vectors to one another, and contouring the diagrams for constant values of Na, Ca, Li, Fe, Mg, Al, Si, and OH. The method is used to depict a wide range of possible tourmaline end-member compositions and solid solutions, starting from a single point. In addition to vector depictions of multicomponent natural tourmalines, vectors are presented for simpler systems such as (Na,Al)-tourmalines, alkali-free tourmalines, and elbaites.

  13. Viral vectors for therapy of neurologic diseases.

    PubMed

    Choudhury, Sourav R; Hudry, Eloise; Maguire, Casey A; Sena-Esteves, Miguel; Breakefield, Xandra O; Grandi, Paola

    2017-07-01

    Neurological disorders - disorders of the brain, spine and associated nerves - are a leading contributor to global disease burden with a shockingly large associated economic cost. Various treatment approaches - pharmaceutical medication, device-based therapy, physiotherapy, surgical intervention, among others - have been explored to alleviate the resulting extent of human suffering. In recent years, gene therapy using viral vectors - encoding a therapeutic gene or inhibitory RNA into a "gutted" viral capsid and supplying it to the nervous system - has emerged as a clinically viable option for therapy of brain disorders. In this Review, we provide an overview of the current state and advances in the field of viral vector-mediated gene therapy for neurological disorders. Vector tools and delivery methods have evolved considerably over recent years, with the goal of providing greater and safer genetic access to the central nervous system. Better etiological understanding of brain disorders has concurrently led to identification of improved therapeutic targets. We focus on the vector technology, as well as preclinical and clinical progress made thus far for brain cancer and various neurodegenerative and neurometabolic disorders, and point out the challenges and limitations that accompany this new medical modality. Finally, we explore the directions that neurological gene therapy is likely to evolve towards in the future. This article is part of the Special Issue entitled "Beyond small molecules for neurological disorders". Copyright © 2016 Elsevier Ltd. All rights reserved.

  14. Adenoassociated virus vectors for genetic immunization.

    PubMed

    Ponnazhagan, Selvarangan

    2002-01-01

    Genetic immunization has initiated a new era of vaccine research, which provides a stable and long-lived source of the protein antigen. Such a vaccine is a simple, robust, and effective means of eliciting both antibody- and cell-mediated immune responses compared with protein or peptide vaccines. Although naked DNA vaccines are relatively simple to produce and handle without significant toxicity or host immunity, those using viral vectors have shown greater efficacy in gene transfer and in inducing both protective and therapeutic immunity in preclinical models. However, clinical translation of results obtained in animal studies with viral vectors has not met with anticipated success so far because of inherent limitations of the vector-associated immunity and antigen specificity. Thus, understanding the requirements for the elicitation of target-specific immunity in host requires that a major cellular arm be unraveled, and modifications of the existing viral vectors and testing of newer ones encompass the technological arm of vaccine research. In this article, I give a comprehensive account of the potential of adenoassociated virus, a nonpathogenic human parvovirus in vaccine development.

  15. Vector ecology of equine piroplasmosis.

    PubMed

    Scoles, Glen A; Ueti, Massaro W

    2015-01-07

    Equine piroplasmosis is a disease of Equidae, including horses, donkeys, mules, and zebras, caused by either of two protozoan parasites, Theileria equi or Babesia caballi. These parasites are biologically transmitted between hosts via tick vectors, and although they have inherent differences they are categorized together because they cause similar pathology and have similar morphologies, life cycles, and vector relationships. To complete their life cycle, these parasites must undergo a complex series of developmental events, including sexual-stage development in their tick vectors. Consequently, ticks are the definitive hosts as well as vectors for these parasites, and the vector relationship is restricted to a few competent tick species. Because the vector relationship is critical to the epidemiology of these parasites, we highlight current knowledge of the vector ecology of these tick-borne equine pathogens, emphasizing tick transmissibility and potential control strategies to prevent their spread.

  16. Targeted radiotherapy with [(90)Y]-SMT 487 in mice bearing human nonsmall cell lung tumor xenografts induced to express human somatostatin receptor subtype 2 with an adenoviral vector.

    PubMed

    Rogers, Buck E; Zinn, Kurt R; Lin, Chin-Yu; Chaudhuri, Tandra R; Buchsbaum, Donald J

    2002-02-15

    Novel approaches to increasing the therapeutic efficacy of targeted radiotherapy of cancer are required. One strategy to achieve this goal is to induce high-level expression of a receptor on the surface of tumor cells that can be targeted with a radiolabeled peptide. The objectives of this study were to 1) induce somatostatin receptor (SSTr2) expression in tumor cells using an adenovirus encoding the SSTr2 gene (AdSSTr2), 2) demonstrate tumor localization of [(111)In]-DTPA-D-Phe(1)-octreotide in AdSSTr2-injected tumors, and 3) show therapeutic efficacy with [(90)Y]-DOTA-D-Phe(1)-Tyr(3)-octreotide ([(90)Y]-SMT 487). SSTr2 expression was validated in vitro by the binding and subsequent internalization of [(111)In]-DTPA-D-Phe(1)-octreotide (21.3% per mg of total protein) in A-427 cells infected with AdSSTr2. In vivo imaging confirmed 5- to 10-fold greater uptake 5.5 hours after intravenous administration of [(111)In]-DTPA-D-Phe(1)-octreotide in AdSSTr2-injected tumors relative to control tumors. For therapy studies, mice bearing established subcutaneous A-427 tumors were given two intratumoral injections of AdSSTr2 1 week apart, followed by an intravenous injection of 400 microCi or 500 microCi [(90)Y]-SMT 487 at 2 and 4 days after each adenoviral administration. Control animals either were not treated or were administered 500 microCi [(90)Y]-SMT 487 with no AdSSTr2 injection. These studies showed that untreated animals and animals treated with no virus and 500 microCi [(90)Y]-SMT 487 had median tumor quadrupling times of 16 and 25 days, respectively. Mice administered AdSSTr2 and either 400 microCi or 500 microCi of [(90)Y]-SMT 487 demonstrated significantly longer median tumor quadrupling times of 40 and 44 days, respectively (P < 0.02). These studies are the first to demonstrate in vivo therapeutic efficacy using a radiolabeled peptide targeted to a receptor expressed on the surface of tumor cells following gene transfer. Future studies will focus on the

  17. Emerging adenoviral vectors for stable correction of genetic disorders.

    PubMed

    Jager, Lorenz; Ehrhardt, Anja

    2007-08-01

    Recent drawbacks in treating patients with severe combined immunodeficiency disorders with retroviral vectors underline the importance of generating novel tools for stable transduction of mammalian cells. Substantial progress has been made over the recent years which may offer important steps towards stable and more importantly safer correction of genetic diseases. This article discusses recent advances for stable transduction of target cells based on adenoviral gene transfer. There is accumulating evidence that recombinant adenoviral vectors (AdVs) based on various human serotypes with a broad cellular tropism and adenoviruses (Ads) from different species will play an important role in future gene therapy applications. In combination with recombinant AdVs for somatic integration these gene transfer vectors offer high transduction efficiencies with potentially safer integration patterns. Other approaches for persistent transgene expression include excision of stable episomes from the adenoviral vector genome, but also long-term persistence of the complete adenoviral vector genome as an episomal DNA molecule was demonstrated and exemplified by the treatment of various genetic diseases in small and large animal models. This review displays advantages but also limitations of these Ad based vector systems. This is the perfect time to pursue such approaches because alternative strategies for stable transduction of mammalian cells undergoing many cell divisions are urgently needed. Looking into the future, we believe that a combination of different components from different viral vectors in concert with non-viral vector systems will be successful in designing significantly optimized transfer vehicles for a broad range of different genetic diseases.

  18. Aerodynamics of thrust vectoring

    NASA Technical Reports Server (NTRS)

    Tseng, J. B.; Lan, C. Edward

    1989-01-01

    Thrust vectoring as a means to enhance maneuverability and aerodynamic performane of a tactical aircraft is discussed. This concept usually involves the installation of a multifunction nozzle. With the nozzle, the engine thrust can be changed in direction without changing the attitude of the aircraft. Change in the direction of thrust induces a significant change in the aerodynamic forces on the aircraft. Therefore, this device can be used for lift-augmenting as well as stability and control purposes. When the thrust is deflected in the longitudinal direction, the lift force and the pitching stability can be manipulated, while the yawing stability can be controlled by directing the thrust in the lateral direction.

  19. Thrust vectoring systems

    NASA Technical Reports Server (NTRS)

    King, H. J.; Schnelker, D.; Ward, J. W.; Dulgeroff, C.; Vahrenkamp, R.

    1972-01-01

    The design, fabrication, and testing of thrust vectorable ion optical systems capable of controlling the thrust direction from both 5- and 30-cm diameter ion thrusters is described. Both systems are capable of greater than 10 deg thrust deflection in any azimuthal direction. The 5-cm system is electrostatic and hence has a short response time and minimal power consumption. It has recently been tested for more than 7500 hours on an operational thruster. The 30-cm system is mechanical, has a response time of the order of 1 min, and consumes less than 0.3% of the total system input power at full deflection angle.

  20. Vector potential photoelectron microscopy.

    PubMed

    Browning, R

    2011-10-01

    A new class of electron microscope has been developed for the chemical microanalysis of a wide range of real world samples using photoelectron spectroscopy. Highly structured, three-dimensional samples, such as fiber mats and fracture surfaces can be imaged, as well as insulators and magnetic materials. The new microscope uses the vector potential field from a solenoid magnet as a spatial reference for imaging. A prototype instrument has demonstrated imaging of uncoated silk, magnetic steel wool, and micron-sized single strand tungsten wires.

  1. Production of lentiviral vectors by transient expression of minimal packaging genes from recombinant adenoviruses.

    PubMed

    Kuate, Seraphin; Stefanou, Daniela; Hoffmann, Dennis; Wildner, Oliver; Uberla, Klaus

    2004-11-01

    The potential of lentiviral vectors for clinical gene therapy has not yet been evaluated. One of the reasons is the cytotoxicity of lentiviral packaging genes which makes the generation of stable producer cell lines difficult. Therefore, a novel packaging system for lentiviral vectors based on transient expression of packaging genes by recombinant adenoviruses was developed. Adenoviral vectors expressing VSV-G, codon-optimized HIV-1 gag-pol, and codon-optimized SIV gag-pol under the control of a tetracycline-regulatable promoter (adenoviral lenti-pack vectors) were constructed and the production levels of this vector system were evaluated. The generated adenoviral lenti-pack vectors could be grown to high titers when transgene expression was suppressed and no evidence for instabilities was obtained. Cells stably transfected with a SIV-based vector construct were converted into lentiviral vector producer cells by infection with the adenoviral lenti-pack vectors. Lentiviral vector titers obtained were as high as vector titers obtained by transient cotransfection experiments. A protocol was developed that allowed preparation of lentiviral vector stocks with undetectable levels of contaminating adenoviral lenti-pack vectors. The adenoviral lenti-pack vectors described should provide a convenient alternative approach to inducible packaging cell lines for large-scale lentiviral vector production. Transient expression of cytotoxic lentiviral packaging genes by the adenoviral lenti-pack vectors circumvents loss of titers during prolonged culture of packaging cell lines. The design of the adenoviral lenti-pack vectors should reduce the risk of transfer of packaging genes to target cells and at the same time provide flexibility with respect to the lentiviral vector constructs that can be packaged.

  2. Engineering the AAV capsid to optimize vector-host-interactions.

    PubMed

    Büning, Hildegard; Huber, Anke; Zhang, Liang; Meumann, Nadja; Hacker, Ulrich

    2015-10-01

    Adeno-associated viral (AAV) vectors are the most widely used delivery system for in vivo gene therapy. Vectors developed from natural AAV isolates achieved clinical benefit for a number of patients suffering from monogenetic disorders. However, high vector doses were required and the presence of pre-existing neutralizing antibodies precluded a number of patients from participation. Further challenges are related to AAV's tropism that lacks cell type selectivity resulting in off-target transduction. Conversely, specific cell types representing important targets for gene therapy like stem cells or endothelial cells show low permissiveness. To overcome these limitations, elegant rational design- as well as directed evolution-based strategies were developed to optimize various steps of AAV's host interaction. These efforts resulted in next generation vectors with enhanced capabilities, that is increased efficiency of cell transduction, targeted transduction of previously non-permissive cell types, escape from antibody neutralization and off-target free in vivo delivery of vector genomes. These important achievements are expected to improve current and pave the way towards novel AAV-based applications in gene therapy and regenerative medicine.

  3. Plasmids with E2 epitope tags: tagging modules for N- and C-terminal PCR-based gene targeting in both budding and fission yeast, and inducible expression vectors for fission yeast.

    PubMed

    Tamm, Tiina

    2009-01-01

    A single-step PCR-based epitope tagging enables fast and efficient gene targeting with various epitope tags. This report presents a series of plasmids for the E2 epitope tagging of proteins in Saccharomyces cerevisiae and Schizosaccharomyces pombe. E2Tags are 10-amino acids (epitope E2a: SSTSSDFRDR)- and 12 amino acids (epitope E2b: GVSSTSSDFRDR)-long peptides derived from the E2 protein of bovine papillomavirus type 1. The modules for C-terminal tagging with E2a and E2b epitopes were constructed by the modification of the pYM-series plasmid. The N-terminal E2a and E2b tagging modules were based on pOM-series plasmid. The pOM-series plasmids were selected for this study because of their use of the Cre-loxP recombination system. The latter enables a marker cassette to be removed after integration into the loci of interest and, thereafter, the tagged protein is expressed under its endogenous promoter. Specifically for fission yeast, high copy pREP plasmids containing the E2a epitope tag as an N-terminal or C-terminal tag were constructed. The properties of E2a and E2b epitopes and the sensitivity of two anti-E2 monoclonal antibodies (5E11 and 3F12) were tested using several S. cerevisiae and Sz. pombe E2-tagged strains.

  4. Electromagnetic couplings of elementary vector particles

    SciTech Connect

    Napsuciale, M.; Rodriguez, S.; Delgado-Acosta, E. G.; Kirchbach, M.

    2008-01-01

    On the basis of the three fundamental principles of (i) Poincare symmetry of space-time, (ii) electromagnetic gauge symmetry, and (iii) unitarity, we construct an universal Lagrangian for the electromagnetic interactions of elementary vector particles, i.e., massive spin-1 particles transforming in the ((1/2),(1/2)) representation space of the homogeneous Lorentz group. We make the point that the first two symmetries alone do not fix the electromagnetic couplings uniquely but solely prescribe a general Lagrangian depending on two free parameters, here denoted by {xi} and g. The first one defines the electric-dipole and the magnetic-quadrupole moments of the vector particle, while the second determines its magnetic-dipole and electric-quadrupole moments. In order to fix the parameters one needs an additional physical input suited for the implementation of the third principle. As such, one chooses Compton scattering off a vector target and requires the cross section to respect the unitarity bounds in the high-energy limit. As a result, we obtain the universal g=2 and {xi}=0 values which completely characterize the electromagnetic couplings of the considered elementary vector field at tree level. The nature of this vector particle, Abelian versus non-Abelian, does not affect this structure. Merely, a partition of the g=2 value into non-Abelian, g{sub na}, and Abelian, g{sub a}=2-g{sub na}, contributions occurs for non-Abelian fields with the size of g{sub na} being determined by the specific non-Abelian group appearing in the theory of interest, be it the standard model or any other theory.

  5. Vector-Mediated In Vivo Antibody Expression.

    PubMed

    Schnepp, Bruce C; Johnson, Philip R

    2014-08-01

    This article focuses on a novel vaccine strategy known as vector-mediated antibody gene transfer, with a particular focus on human immunodeficiency virus (HIV). This strategy provides a solution to the problem of current vaccines that fail to generate neutralizing antibodies to prevent HIV-1 infection and AIDS. Antibody gene transfer allows for predetermination of antibody affinity and specificity prior to "immunization" and avoids the need for an active humoral immune response against the HIV envelope protein. This approach uses recombinant adeno-associated viral (rAAV) vectors, which have been shown to transduce muscle with high efficiency and direct the long-term expression of a variety of transgenes, to deliver the gene encoding a broadly neutralizing antibody into the muscle. Following rAAV vector gene delivery, the broadly neutralizing antibodies are endogenously synthesized in myofibers and passively distributed to the circulatory system. This is an improvement over classical passive immunization strategies that administer antibody proteins to the host to provide protection from infection. Vector-mediated gene transfer studies in mice and monkeys with anti-HIV and simian immunodeficiency virus (SIV)-neutralizing antibodies demonstrated long-lasting neutralizing activity in serum with complete protection against intravenous challenge with virulent HIV and SIV. These results indicate that existing potent anti-HIV antibodies can be rapidly moved into the clinic. However, this methodology need not be confined to HIV. The general strategy of vector-mediated antibody gene transfer can be applied to other difficult vaccine targets such as hepatitis C virus, malaria, respiratory syncytial virus, and tuberculosis.

  6. Water-solubilizing Hydrophobic ZnAgInSe/ZnS QDs with Tumor-targeted cRGD-Sulfobetaine-PIMA-Histamine Ligands via a Self-assembly Strategy for Bio-Imaging.

    PubMed

    Deng, Tao; Peng, Yanan; Zhang, Rong; Wang, Jie; Zhang, Jie; Gu, Yue-Qing; Huang, Dechun; Deng, Dawei

    2017-03-15

    Exploring the organic-to-aqueous phase transfer of quantum dots (QDs) is significant for achieving their versatile applications in biomedαical fields. In this thematic issue, surface modification, size control and biocompatibility of QDs and QDs-based nanocomposites are core problems. Herein, the new highly fluorescent tumor-targeted QDs-clusters consisting of ZnAgInSe/ZnS (ZAISe/ZnS) QDs and sulfobetaine-PIMA-histamine (SPH) polymer with the ανβ3 integrin receptor cyclic RGD (c-RGD) were developed via ligand exchange and an accompanying self-assemble process. It was found that the structure of RGD-SPH QDs-clusters was propitious to reduce the capture of reticulo-endothelial system (RES) in virtue of external stealth ligands, and benefit to selectively accumulate at the tumor site after intravenous injection via active tumor targeting cooperated with the enhanced permeability and retention (EPR) effect. In the meantime, those clusters also recognized and enriched to cell surface when co-cultured with the ανβ3 integrin receptor overexpressed malignant cells (U87MG tumors). Based on the results, fabricating mutil-functional nanocomposites integrated with the long-term circulation and dual-targeting effects should be an interesting strategy for imaging cancer in vitro and in vivo.

  7. Targeted theranostic platinum(IV) prodrug with a built-in aggregation-induced emission light-up apoptosis sensor for noninvasive early evaluation of its therapeutic responses in situ.

    PubMed

    Yuan, Youyong; Kwok, Ryan T K; Tang, Ben Zhong; Liu, Bin

    2014-02-12

    Targeted drug delivery to tumor cells with minimized side effects and real-time in situ monitoring of drug efficacy is highly desirable for personalized medicine. In this work, we report the synthesis and biological evaluation of a chemotherapeutic Pt(IV) prodrug whose two axial positions are functionalized with a cyclic arginine-glycine-aspartic acid (cRGD) tripeptide for targeting integrin αvβ3 overexpressed cancer cells and an apoptosis sensor which is composed of tetraphenylsilole (TPS) fluorophore with aggregation-induced emission (AIE) characteristics and a caspase-3 enzyme specific Asp-Glu-Val-Asp (DEVD) peptide. The targeted Pt(IV) prodrug can selectively bind to αvβ3 integrin overexpressed cancer cells to facilitate cellular uptake. In addition, the Pt(IV) prodrug can be reduced to active Pt(II) drug in cells and release the apoptosis sensor TPS-DEVD simultaneously. The reduced Pt(II) drug can induce the cell apoptosis and activate caspase-3 enzyme to cleave the DEVD peptide sequence. Due to free rotation of the phenylene rings, TPS-DEVD is nonemissive in aqueous media. The specific cleavage of DEVD by caspase-3 generates the hydrophobic TPS residue, which tends to aggregate, resulting in restriction of intramolecular rotations of the phenyl rings and ultimately leading to fluorescence enhancement. Such noninvasive and real-time imaging of drug-induced apoptosis in situ can be used as an indicator for early evaluation of the therapeutic responses of a specific anticancer drug.

  8. Poxviral vectors for cancer immunotherapy

    PubMed Central

    Kim, Joseph W.; Gulley, James L.

    2012-01-01

    Introduction Poxviral vaccines have been given to over 1 billion people in the successful global eradication of smallpox. Since then, recombinant poxviruses have been investigated extensively as a novel immunotherapy for cancer, undergoing several iterations to optimize their immunogenicity and efficacy. The current platform expressing multiple costimulatory molecules plus a tumor-associated antigen such as PSA, i.e., PSA-TRICOM (PROSTVAC-V/F), is promising and is currently in a phase III randomized, placebo-controlled clinical trial in metastatic castration-resistant prostate cancer. Areas covered This review discusses the clinical development of poxviral-based cancer vaccines, with a particular focus on the rationale for combining vaccines with other treatment modalities, including radiotherapy, chemotherapy, hormonal therapy, other immune-based therapies, and molecularly targeted therapy. We also discuss the importance of appropriate patient selection in clinical trial design. Expert Opinion Preclinical and early clinical studies with poxviral vector vaccines have shown promising results with this novel immunologic approach both as vaccine alone and combined with other therapies. The challenges of translating the science of immunotherapy to clinical practice include clinical trial design that includes appropriate patient selection, appropriate endpoints, and identification of meaningful surrogate biomarkers. PMID:22413824

  9. Extended vector-tensor theories

    NASA Astrophysics Data System (ADS)

    Kimura, Rampei; Naruko, Atsushi; Yoshida, Daisuke

    2017-01-01

    Recently, several extensions of massive vector theory in curved space-time have been proposed in many literatures. In this paper, we consider the most general vector-tensor theories that contain up to two derivatives with respect to metric and vector field. By imposing a degeneracy condition of the Lagrangian in the context of ADM decomposition of space-time to eliminate an unwanted mode, we construct a new class of massive vector theories where five degrees of freedom can propagate, corresponding to three for massive vector modes and two for massless tensor modes. We find that the generalized Proca and the beyond generalized Proca theories up to the quartic Lagrangian, which should be included in this formulation, are degenerate theories even in curved space-time. Finally, introducing new metric and vector field transformations, we investigate the properties of thus obtained theories under such transformations.

  10. Hyperbolic-symmetry vector fields.

    PubMed

    Gao, Xu-Zhen; Pan, Yue; Cai, Meng-Qiang; Li, Yongnan; Tu, Chenghou; Wang, Hui-Tian

    2015-12-14

    We present and construct a new kind of orthogonal coordinate system, hyperbolic coordinate system. We present and design a new kind of local linearly polarized vector fields, which is defined as the hyperbolic-symmetry vector fields because the points with the same polarization form a series of hyperbolae. We experimentally demonstrate the generation of such a kind of hyperbolic-symmetry vector optical fields. In particular, we also study the modified hyperbolic-symmetry vector optical fields with the twofold and fourfold symmetric states of polarization when introducing the mirror symmetry. The tight focusing behaviors of these vector fields are also investigated. In addition, we also fabricate micro-structures on the K9 glass surfaces by several tightly focused (modified) hyperbolic-symmetry vector fields patterns, which demonstrate that the simulated tightly focused fields are in good agreement with the fabricated micro-structures.

  11. A fast vector array adaptive beam forming method

    NASA Astrophysics Data System (ADS)

    Li, Zhizhong; Chen, Zhe; Li, Haitao; Xu, Zhongliang

    2017-06-01

    Based on model features of the vector sensor array signals, the paper transforms the time delay of broadband signals in time domain into the phase shift of different sub-bands in frequency domain to realize accurate time delay, and uses Hilbert Transform to construct analytic signals to form a fast vector array adaptive beam forming algorithm flow. The verification result with experimental data shows that this algorithm has much better target resolution capability than conventional beam forming algorithm. With the increase of 4-6dB in target detection capability, it has bright application prospect.

  12. Insecticide resistance in disease vectors from Mayotte: an opportunity for integrated vector management

    PubMed Central

    2014-01-01

    Background Mayotte, a small island in the Indian Ocean, has been affected for many years by vector-borne diseases. Malaria, Bancroftian filariasis, dengue, chikungunya and Rift Valley fever have circulated or still circulate on the island. They are all transmitted by Culicidae mosquitoes. To limit the impact of these diseases on human health, vector control has been implemented for more than 60 years on Mayotte. In this study, we assessed the resistance levels of four major vector species (Anopheles gambiae, Culex pipiens quinquefasciatus, Aedes aegypti and Aedes albopictus) to two types of insecticides: i) the locally currently-used insecticides (organophosphates, pyrethroids) and ii) alternative molecules that are promising for vector control and come from different insecticide families (bacterial toxins or insect growth regulators). When some resistance was found to one of these insecticides, we characterized the mechanisms involved. Methods Larval and adult bioassays were used to evaluate the level of resistance. When resistance was found, we tested for the presence of metabolic resistance through detoxifying enzyme activity assays, or for target-site mutations through molecular identification of known resistance alleles. Results Resistance to currently-used insecticides varied greatly between the four vector species. While no resistance to any insecticides was found in the two Aedes species, bioassays confirmed multiple resistance in Cx. p. quinquefasciatus (temephos: ~ 20 fold and deltamethrin: only 10% mortality after 24 hours). In An. gambiae, resistance was scarce: only a moderate resistance to temephos was found (~5 fold). This resistance appears to be due only to carboxyl-esterase overexpression and not to target modification. Finally, and comfortingly, none of the four species showed resistance to any of the new insecticides. Conclusions The low resistance observed in Mayotte’s main disease vectors is particularly interesting, because it leaves a

  13. Molecular Engineering of Vector-Based Oncolytic and Imaging Approaches for Advanced Prostate Cancer

    DTIC Science & Technology

    2007-02-01

    have incorporated a highly potent and prostate-specific transcriptional regulatory system (TSTA) in adenoviral vectors such that the expression of...engineering, we have demonstrated the feasibility and the functionality of the prostate-targeted oncolytic system . The immediate future plan is to apply...step transcriptional amplification (TSTA) system to mediate prostate- targeted gene expression. In diagnostic applications, this targeted vector will be

  14. An adaptive vector quantization scheme

    NASA Technical Reports Server (NTRS)

    Cheung, K.-M.

    1990-01-01

    Vector quantization is known to be an effective compression scheme to achieve a low bit rate so as to minimize communication channel bandwidth and also to reduce digital memory storage while maintaining the necessary fidelity of the data. However, the large number of computations required in vector quantizers has been a handicap in using vector quantization for low-rate source coding. An adaptive vector quantization algorithm is introduced that is inherently suitable for simple hardware implementation because it has a simple architecture. It allows fast encoding and decoding because it requires only addition and subtraction operations.

  15. Multistage vector (MSV) therapeutics.

    PubMed

    Wolfram, Joy; Shen, Haifa; Ferrari, Mauro

    2015-12-10

    One of the greatest challenges in the field of medicine is obtaining controlled distribution of systemically administered therapeutic agents within the body. Indeed, biological barriers such as physical compartmentalization, pressure gradients, and excretion pathways adversely affect localized delivery of drugs to pathological tissue. The diverse nature of these barriers requires the use of multifunctional drug delivery vehicles that can overcome a wide range of sequential obstacles. In this review, we explore the role of multifunctionality in nanomedicine by primarily focusing on multistage vectors (MSVs). The MSV is an example of a promising therapeutic platform that incorporates several components, including a microparticle, nanoparticles, and small molecules. In particular, these components are activated in a sequential manner in order to successively address transport barriers. Copyright © 2015 Elsevier B.V. All rights reserved.

  16. The MSFC vector magnetograph

    NASA Technical Reports Server (NTRS)

    Hagyard, M. J.; Cumings, N. P.; West, E. A.

    1981-01-01

    The NASA/Marshall Space Flight Center's solar vector magnetograph system allows measurements of all components of the Sun's photospheric magnetic field over a 5 x 5 or 2.5 x 2.5 arc min square field of view with an optimum time resolution of approximately 100 sec and an optimum signal-to-noise of approximately 1000. The basic system components are described, including the optics, detector, digital system, and associated electronics. Automatic sequencing and control functions are outlined as well as manual selections of system parameters which afford unique system flexibility. Results of system calibration and performance are presented, including linearity, dynamic range, uniformity, spatial and spectral resolutions, signal-to-noise, electro-optical retardation and polarization calibration.

  17. Chameleon vector bosons

    SciTech Connect

    Nelson, Ann E.

    2008-05-01

    We show that for a force mediated by a vector particle coupled to a conserved U(1) charge, the apparent range and strength can depend on the size and density of the source, and the proximity to other sources. This chameleon effect is due to screening from a light charged scalar. Such screening can weaken astrophysical constraints on new gauge bosons. As an example we consider the constraints on chameleonic gauged B-L. We show that although Casimir measurements greatly constrain any B-L force much stronger than gravity with range longer than 0.1 {mu}m, there remains an experimental window for a long-range chameleonic B-L force. Such a force could be much stronger than gravity, and long or infinite range in vacuum, but have an effective range near the surface of the earth which is less than a micron.

  18. Entangled vector vortex beams

    NASA Astrophysics Data System (ADS)

    D'Ambrosio, Vincenzo; Carvacho, Gonzalo; Graffitti, Francesco; Vitelli, Chiara; Piccirillo, Bruno; Marrucci, Lorenzo; Sciarrino, Fabio

    2016-09-01

    Light beams having a vectorial field structure, or polarization, that varies over the transverse profile and a central optical singularity are called vector vortex (VV) beams and may exhibit specific properties such as focusing into "light needles" or rotation invariance. VV beams have already found applications in areas ranging from microscopy to metrology, optical trapping, nano-optics, and quantum communication. Individual photons in such beams exhibit a form of single-particle quantum entanglement between different degrees of freedom. On the other hand, the quantum states of two photons can be also entangled with each other. Here, we combine these two concepts and demonstrate the generation of quantum entanglement between two photons that are both in VV states: a form of entanglement between two complex vectorial fields. This result may lead to quantum-enhanced applications of VV beams as well as to quantum information protocols fully exploiting the vectorial features of light.

  19. Multistage vector (MSV) therapeutics

    PubMed Central

    Wolfram, Joy; Shen, Haifa; Ferrari, Mauro

    2015-01-01

    One of the greatest challenges in the field of medicine is obtaining controlled distribution of systemically administered therapeutic agents within the body. Indeed, biological barriers such as physical compartmentalization, pressure gradients, and excretion pathways adversely affect localized delivery of drugs to pathological tissue. The diverse nature of these barriers requires the use of multifunctional drug delivery vehicles that can overcome a wide range of sequential obstacles. In this review, we explore the role of multifunctionality in nanomedicine by primarily focusing on multistage vectors (MSVs). The MSV is an example of a promising therapeutic platform that incorporates several components, including a microparticle, nanoparticles, and small molecules. In particular, these components are activated in a sequential manner in order to successively address transport barriers. PMID:26264836

  20. Solar imaging vector magnetograph

    NASA Technical Reports Server (NTRS)

    Canfield, Richard C.

    1993-01-01

    This report describes an instrument which has been constructed at the University of Hawaii to make observations of the magnetic field in solar active regions. Detailed knowledge of active region magnetic structures is crucial to understanding many solar phenomena, because the magnetic field both defines the morphology of structures seen in the solar atmosphere and is the apparent energy source for solar flares. The new vector magnetograph was conceived in response to a perceived discrepancy between the capabilities of X ray imaging telescopes to be operating during the current solar maximum and those of existing magnetographs. There were no space-based magnetographs planned for this period; the existing ground-based instruments variously suffered from lack of sensitivity, poor time resolution, inadequate spatial resolution or unreliable sites. Yet the studies of flares and their relationship to the solar corona planned for the 1991-1994 maximum absolutely required high quality vec