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pBABEpuro_uPA Retroviral Vector

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[#RTV-501] pBABEpuro_uPA Retroviral Vector

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(1) Efficient CRISPR/Cas9-Mediated Mutagenesis in Primary Murine T Lymphocytes.[TOP]

Pubmed ID :30312021
Publication Date : //
The ability to alter gene expression directly in T lymphocytes has provided a powerful tool for understanding T cell biology, signaling, and function. Manipulation of T cell clones and primary T cells has been accomplished primarily through overexpression or gene-silencing studies using cDNAs or shRNAs, respectively, which are often delivered by retroviral or lentiviral transduction or direct transfection methods. The recent development of CRISPR/Cas9-based mutagenesis has revolutionized genomic editing, allowing unprecedented genetic manipulation of many cell types with greater precision and ease. This article outlines a protocol for CRISPR/Cas9-mediated mutagenesis in primary T lymphocytes from Cas9 transgenic mice using retroviral delivery of guide RNAs. © 2018 by John Wiley & Sons, Inc.

Authors : Huang Bonnie, Johansen Kristoffer Haurum, Schwartzberg Pamela L,



(2) Avian Bioreactor Systems: A Review.[TOP]

Pubmed ID :30306403
Publication Date : //
Animal bioreactors are genetically modified animal systems that have the potential to reduce production cost, and improve production efficiency, of pharmaceutically relevant recombinant proteins. Several species including goats, cattle, rabbits, and avians have been genetically modified to secrete target proteins into milk, egg whites, blood, or other bodily fluids. There are several advantages associated with the use of avians as bioreactor systems. Avians have a short generation time, leading to the quick establishment of a transgenic line and high egg production. Transgenic avian systems allow for appropriate post-translational modification, as opposed to prokaryotic cell culture bioreactors, and have higher productivity than mammalian cell culture systems. Furthermore, recombinant proteins can be incorporated into egg whites and easily collected from the sterile environment of the egg. Magnum-specific expression of target genes has been achieved by use of the ovalbumin promoter, leading to a localization of the target protein into the avian egg. In this review, we discuss the current advancements, future potential, and limitations of avian bioreactor systems.

Authors : Woodfint Rachel M, Hamlin Erin, Lee Kichoon,



(3) Loss of Gadd45b accelerates BCR-ABL-driven CML.[TOP]

Pubmed ID :30279966
Publication Date : //
Gadd45b is a member of Gadd45 stress sensor protein family that also includes Gadd45a & Gadd45g. To investigate the effect of Gadd45b in bcr-abl oncogene driven chronic myeloid leukemia (CML) development, syngeneic wild type lethally irradiated mice were reconstituted with either wild type or Gadd45b null myeloid progenitors transduced with a retroviral vector expressing BCR-ABL. Loss of Gadd45b was observed to accelerate BCR-ABL driven CML development with shortened median mouse survival time. BCR-ABL Gadd45b deficient CML progenitors exhibited increased proliferation and decreased apoptosis, associated with hyper-activation of c-Jun NH-terminal kinase and Stat5. These results provide novel evidence that gadd45b, like gadd45a, functions as a suppressor of BCR-ABL driven leukemia, albeit via a different mechanism.

Authors : Sha Xiaojin, Hoffman Barbara, Liebermann Dan A,



(4) NOTCH1 regulates the viability of cholangiocarcinoma cells via 14-3-3 theta.[TOP]

Pubmed ID :30264361
Publication Date : //
Notch signaling has been reported to correlate with tumor progression and metastasis in several types of cancer. In cholangiocarcinoma (CCA), it has recently been shown that NOTCH1 is overexpressed in both nucleus and cytoplasm of CCA cells; however, the complete understanding of Notch signaling in CCA is still lacking. Here, we aimed to understand the functions of NOTCH1 in CCA cells and the molecular mechanisms that underlie those functions. We used retroviral vectors to overexpress active forms of NOTCH1, the NOTCH1 intracellular domain (N1ICD) and N1ICD that lacks the RBP-J-associated module (RAM), in human CCA cell lines RMCCA-1 and HuCCA-1. Our results showed that activation of Notch signaling by both N1ICD variants enhanced CCA cell proliferation and survival via upregulation of pro-survival protein Mcl-1 and Bcl-xL. Moreover, our LC-MS/MS proteomic studies demonstrated that NOTCH1 may cooperate with 14-3-3 theta to promote CCA cell survival. Knockdown of 14-3-3 theta in RMCCA-1 cells overexpressing N1ICD, diminished pro-survival effects of N1ICD under gemcitabine treatment. In conclusion, these data demonstrated that NOTCH1 plays a role in CCA cell proliferation and survival via the regulation of 14-3-3 theta in a RAM-independent fashion.

Authors : Singrang Nongnuch, Kittisenachai Suthathip, Roytrakul Sittiruk, Svasti Jisnuson, Kangsamaksin Thaned,



(5) The Journey of Virus Engineered Dendritic Cells From Bench to Bedside: A Bumpy Road.[TOP]

Pubmed ID :30254636
Publication Date : //
Dendritic cells (DCs) are recognized as highly potent antigen-presenting cells that are able to stimulate cytotoxic T lymphocyte (CTL) responses with antitumor activity. Consequently, DCs have been explored as cellular vaccines in cancer immunotherapy. To that end, DCs are modified with tumor antigens to enable presentation of antigen-derived peptides to CTLs. In this review we discuss the use of viral vectors for modification of DCs, focusing on their clinical applications as anticancer vaccines. Among the viral vectors discussed are those derived from viruses belonging to the families of the , and . We will further shed light on how the combination of viral vector-based vaccination with T-cell supporting strategies will bring this strategy to the next level.

Authors : Goyvaerts Cleo, Breckpot Karine,



(6) JAK3 mutations and HOXA9 expression are important cooperating events in T-cell acute lymphoblastic leukemia.[TOP]

Pubmed ID :30250904
Publication Date : //
Sequencing data from large cohorts of T-cell acute lymphoblastic leukemia patients identified a significant association between the presence of mutations and ectopic expression. Mouse models using a constitutive or novel inducible retroviral expression vector to express the mutant and led to the development of an aggressive leukemia in vivo, with shorter latency than or alone. This was primarily due to the co-binding of STAT5 and HOXA9 to the same genomic loci leading to increased oncogenic JAK-STAT signaling.

Authors : de Bock Charles E, Cools Jan,



(7) Retrovirus-Mediated Transfection of the Tissue-type Plasminogen Activator Gene Results in Increased Thrombolysis of Blood Clots.[TOP]

Pubmed ID :30218285
Publication Date : //
Tissue-type plasminogen activator (tPA) is involved in the lysis of blood clots. In this study, we attempted to target thrombolysis and enhance blood clot lysis by generating a construct (pLEGFP-N1-tPA) to integrate tPA gene into the genome of different cell lines. pLEGFP-N1-tPA construct was generated and used to target the tPA gene in different cell lines. The thrombolytic effects mediated by the supernatant from transfected HeLa cells and Linx cells were assessed using plasma thrombus plates. Furthermore, enhanced green fluorescent protein (EGFP), which was fused to the tPA gene in the pLEGFP-N1-tPA construct, was analyzed under the fluorescent microscope to assess tPA localization. We also monitored tPA activity and expression in the transfected cell lines. As part of the study, we successfully generated the pLEGFP-N1-tPA construct. The sequence of this construct was verified and the construct was subsequently used to generate the PT67/pLEGFP-N1-tPA cell line. The pLEGFP-N1-tPA construct was also used to transfect HeLa cells and Linx cells. We observed that supernatants from transfected cells were capable of lysing thrombi. In addition, tPA activity and tPA concentration were elevated in the latter supernatants and tPA was rapidly and stably expressed in the transfected cell lines. These results reveal a potentially important thrombolytic role for tPA-targeted gene therapy following cardiac valve replacement.

Authors : Wu Xianhua, Gong Yongsheng, Ding Xuebing, Cheng Gang, Yan Weiya, She Xiaowei, Wang Changxing, Li Xia,



(8) Screening Clinical Cell Products for Replication Competent Retrovirus: The National Gene Vector Biorepository Experience.[TOP]

Pubmed ID :30211249
Publication Date : //
Replication-competent retrovirus (RCR) is a safety concern for individuals treated with retroviral gene therapy. RCR detection assays are used to detect RCR in manufactured vector, transduced cell products infused into research subjects, and in the research subjects after treatment. In this study, we reviewed 286 control (n = 4) and transduced cell products (n = 282) screened for RCR in the National Gene Vector Biorepository. The transduced cell samples were submitted from 14 clinical trials. All vector products were previously shown to be negative for RCR prior to use in cell transduction. After transduction, all 282 transduced cell products were negative for RCR. In addition, 241 of the clinical trial participants were also screened for RCR by analyzing peripheral blood at least 1 month after infusion, all of which were also negative for evidence of RCR infection. The majority of vector products used in the clinical trials were generated in the PG13 packaging cell line. The findings suggest that screening of the retroviral vector product generated in PG13 cell line may be sufficient and that further screening of transduced cells does not provide added value.

Authors : Cornetta Kenneth, Duffy Lisa, Feldman Steven A, Mackall Crystal L, Davila Marco L, Curran Kevin J, Junghans Richard P, Tang Jean Yuh, Kochenderfer James N, O'Cearbhaill Roisin, Archer Gary, Kiem Hans-Peter, Shah Nirali N, Delbrook Cindy, Kaplan Rosie, Brentjens Renier J, Rivière Isabelle, Sadelain Michel, Rosenberg Steven A,



(9) Novel Human NK Cell Line Carrying CAR Targeting EGFRvIII Induces Antitumor Effects in Glioblastoma Cells.[TOP]

Pubmed ID :30194149
Publication Date : //
Natural killer (NK) cells are considered potential antitumor effector cells. The aim of this study was to establish a novel type of a chimeric antigen receptor (CAR) NK cell line (CAR-KHYG-1) specific for epidermal growth factor receptor variant III (EGFRvIII)-expressing tumors and investigate the anti-tumor activity of EGFRvIII-specific-CAR-KHYG-1 (EvCAR-KHYG-1).

Authors : Murakami Toshiharu, Nakazawa Tsutomu, Natsume Atsushi, Nishimura Fumihiko, Nakamura Mitsutoshi, Matsuda Ryosuke, Omoto Koji, Tanaka Yoshitaka, Shida Youichi, Park Young-Soo, Motoyama Yasushi, Nakagawa Ichiro, Yamada Shuichi, Tamura Kentaro, Takeshima Yasuhiro, Takamura Yoshiaki, Wakabayashi Toshihiko, Nakase Hiroyuki,



(10) Thymus autonomy as a prelude to leukemia.[TOP]

Pubmed ID :30187694
Publication Date : //
Cell competition in the thymus promotes turnover and functions as a tumor suppressor by inhibiting leukemia. Using thymus transplantation experiments, we have shown that the presence of T lymphocyte precursors, recently seeding the thymus, promotes the clearance of precursors with a longer time of thymus residency. If cell competition is impaired and no cells seed the thymus, the organ is capable of sustaining T lymphocyte production, a state termed thymus autonomy. However, we observed consistently that prolonged autonomy is permissive to the emergence of T cell acute lymphoblastic leukemia (T-ALL). This resembled the onset of T-ALL in patients treated by gene therapy for X-linked severe combined immunodeficiency (SCID-X1). Following treatment, thymus activity was established, with T lymphocyte production, although no bone marrow contribution was detected. However, some patients developed T-ALL. The favored explanation for malignant transformation was considered to be genotoxicity due to integration of the retroviral vector next to oncogenes, thereby activating them ectopically. Although plausible, we consider an alternative, mutually nonexclusive explanation: that any condition enabling prolonged thymus autonomy will promote leukemogenesis. In support of this view, two independent studies have recently shown that the efficacy of reconstitution of the bone marrow in the context of SCID-X1 dramatically influences the outcome of treatment, and that lymphoid malignancies emerge following transplantation of a small number of healthy progenitors. Here, we discuss the most recent data in light of our own studies in thymopoiesis and the conditions that trigger malignant transformation of thymocytes in various experimental and clinical settings.

Authors : Paiva Rafael A, Ramos Camila V, Martins Vera C,