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pBABEpuro_c_Abl_TM Retroviral Vector

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[#RTV-403] pBABEpuro_c_Abl_TM Retroviral Vector


RTV-403 | pBABEpuro_c_Abl_TM Retroviral Vector, 100 µL
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(1) Sustained and regulated gene expression by Tet-inducible "all-in-one" retroviral vectors containing the HNRPA2B1-CBX3 UCOE.[TOP]

Pubmed ID :30508767
Publication Date : //
Genetic modification of induced pluripotent stem (iPS) cells may be necessary for the generation of effector cells for cellular therapies. Hereby, it can be important to induce transgene expression at restricted and defined time windows, especially if it interferes with pluripotency or differentiation. To achieve this, inducible expression systems can be used such as the tetracycline-inducible retroviral vector system, however, retroviral expression can be subjected to epigenetic silencing or to position-effect variegation. One strategy to overcome this is the incorporation of ubiquitous chromatin opening elements (UCOE's) into retroviral vectors to maintain a transcriptionally permissive chromatin state at the integration site. In this study, we developed Tet-inducible all-in-one gammaretroviral vectors carrying different sized UCOE's derived from the A2UCOE. The ability to prevent vector silencing by preserving the Tet-regulatory potential was investigated in different cell lines, and in murine and human iPS cells. A 670-bp fragment spanning the CBX3 promoter region of A2UCOE (U670) was the most potent element in preventing silencing, and conferred the strongest expression from the vector in the induced state. While longer fragments of A2UCOEs also sustained expression, vector titers and induction efficiencies were impaired. Finally, we demonstrate that U670 can be used for constitutive expression of the transactivator in the all-in-one vector for faithful regulation of transgenes by doxycycline, including the thrombopoietin receptor Mpl conferring cytokine-dependent cell growth.

Authors : Cullmann Katharina, Blokland Kaj E C, Sebe Attila, Schenk Franziska, Ivics Zoltán, Heinz Niels, Modlich Ute,

(2) HIV-1 T cell epitopes targeted to Rhesus macaque CD40 and DCIR: A comparative study of prototype dendritic cell targeting therapeutic vaccine candidates.[TOP]

Pubmed ID :30500852
Publication Date : //
HIV-1 infection can be controlled by anti-retroviral drug therapy, but this is a lifetime treatment and the virus remains latent and rapidly rebounds if therapy is stopped. HIV-1-infected individuals under this drug regimen have increased rates of cancers, cardiovascular diseases, and autoimmunity due to compromised immunity. A therapeutic vaccine boosting cellular immunity against HIV-1 is therefore desirable and, possibly combined with other immune modulating agents, could obviate the need for long-term drug therapies. An approach to elicit strong T cell-based immunity is to direct virus protein antigens specifically to dendritic cells (DCs), which are the key cell type for controlling immune responses. For eliciting therapeutic cellular immunity in HIV-1-infected individuals, we developed vaccines comprised of five T cell epitope-rich regions of HIV-1 Gag, Nef, and Pol (HIV5pep) fused to monoclonal antibodies that bind either, the antigen presenting cell activating receptor CD40, or the endocytic dendritic cell immunoreceptor DCIR. The study aimed to demonstrate vaccine safety, establish efficacy for broad T cell responses in both primed and naïve settings, and identify one candidate vaccine for human therapeutic development. The vaccines were administered to Rhesus macaques by intradermal injection with poly-ICLC adjuvant. The animals were either i) naïve or, ii) previously primed with modified vaccinia Ankara vector (MVA) encoding HIV-1 Gag, Pol, and Nef (MVA GagPolNef). In the MVA-primed groups, both DC-targeting vaccinations boosted HIV5pep-specific blood CD4+ T cells producing multiple cytokines, but did not affect the MVA-elicited CD8+ T cell responses. In the naive groups, both DC-targeting vaccines elicited antigen-specific polyfunctional CD4+ and CD8+ T cell responses to multiple epitopes and these responses were unchanged by a subsequent MVA GagPolNef boost. In both settings, the T cell responses elicited via the CD40-targeting vaccine were more robust and were detectable in all the animals, favoring further development of the CD40-targeting vaccine for therapeutic vaccination of HIV-1-infected individuals.

Authors : Flamar Anne-Laure, Bonnabau Henri, Zurawski Sandra, Lacabaratz Christine, Montes Monica, Richert Laura, Wiedemann Aurelie, Galmin Lindsey, Weiss Deborah, Cristillo Anthony, Hudacik Lauren, Salazar Andres, Peltekian Cécile, Thiebaut Rodolphe, Zurawski Gerard, Levy Yves,

(3) Early T follicular helper cell responses and germinal center reactions are associated with viremia control in immunized Rhesus macaques.[TOP]

Pubmed ID :30463978
Publication Date : //
T follicular helper (T) cells are fundamental in germinal center (GC) maturation and selection of antigen-specific B cells within secondary lymphoid organs. GC-resident T cells have been fully characterized in human HIV infection. However, the role of GC T cells in GC B cell responses following various SIV vaccine regimens in Rhesus macaques (RMs) has not been fully investigated. We characterized GC T cells of RMs over the course of a mucosal/systemic vaccination regimen to elucidate GC formation and SIV humoral response generation. Animals were mucosally primed twice with replicating Ad5hr-SIV recombinants and systemically boosted with ALVAC/Env or DNA&Env including SIV gp120 proteins. Lymph nodes were biopsied in macaque subgroups pre-vaccination and at days 3, 7, or 14 after the 2 Ad5hr-SIV prime and the 2 vector/Env boost. Evaluations of GC T and GC B cell dynamics including correlation analyses supported a significant role of early GC T cells in providing B cell help during initial phases of GC formation. GC T responses at day 3 post-mucosal priming were consistent with generation of Env-specific memory B cells in GCs and elicitation of prolonged Env-specific humoral immunity in the rectal mucosa. GC Env-specific memory B cell responses elicited early post-systemic boosting correlated significantly with decreased viremia post-infection. Our results highlight the importance of early GC T cell responses for robust GC maturation and generation of long-lasting SIV specific humoral responses at mucosal and systemic sites. Further investigation of GC T cell dynamics should facilitate development of an efficacious HIV vaccine. The modest HIV protection observed in the human RV144 vaccine trial associated antibody responses with vaccine efficacy. T follicular helper (T) cells are CD4 T cells that select antibody secreting cells with high antigenic affinity in germinal centers (GCs) within secondary lymphoid organs. To evaluate the role of T cells in eliciting prolonged viral-specific humoral responses, we vaccinated Rhesus macaques with a combined mucosal prime/systemic boost regimen followed by repeated low-dose intrarectal challenges with SIV mimicking human exposure to HIV-1. Although the vaccine regimen did not prevent SIV infection, decreased viremia was observed in the immunized macaques. Importantly, vaccine-induced T responses elicited at day 3 post-immunization and robust GC maturation were strongly associated. Further, early T-dependent SIV-specific B cell responses were also correlated with decreased viremia. Our findings highlight the contribution of early vaccine-induced GC T responses to elicitation of SIV-specific humoral immunity and implicate their participation in SIV control.

Authors : Helmold Hait Sabrina, Vargas-Inchaustegui Diego A, Musich Thomas, Mohanram Venkatramanan, Tuero Iskra, Venzon David J, Bear Jenifer, Rosati Margherita, Vaccari Monica, Franchini Genoveffa, Felber Barbara K, Pavlakis George N, Robert-Guroff Marjorie,

(4) Role of interleukin-7 in fusion of rat bone marrow mesenchymal stem cells with cardiomyocytes in vitro and improvement of cardiac function in vivo.[TOP]

Pubmed ID :30451388
Publication Date : //
Mesenchymal stem cells (MSCs) hold significant promise as potential therapeutic candidates following cardiac injury. However, to ensure survival of transplanted cells in ischemic environment, it is beneficial to precondition them with growth factors that play important role in cell survival and proliferation. Aim of this study is to use interleukin-7 (IL-7), a cell survival growth factor, to enhance the potential of rat bone marrow MSCs in terms of cell fusion in vitro and cardiac function in vivo.

Authors : Haneef Kanwal, Ali Anwar, Khan Irfan, Naeem Nadia, Jamall Siddiqua, Salim Asmat,

(5) Post-mitotic BET-induced reshaping of integrase quaternary structure supports wild-type MLV integration.[TOP]

Pubmed ID :30445610
Publication Date : //
The Moloney murine leukemia virus (MLV) is a prototype gammaretrovirus requiring nuclear disassembly before DNA integration. In the nucleus, integration site selection towards promoter/enhancer elements is mediated by the host factor bromo- and extraterminal domain (BET) proteins (bromodomain (Brd) proteins 2, 3 and 4). MLV-based retroviral vectors are used in gene therapy trials. In some trials leukemia occurred through integration of the MLV vector in close proximity to cellular oncogenes. BET-mediated integration is poorly understood and the nature of integrase oligomers heavily debated. Here, we created wild-type infectious MLV vectors natively incorporating fluorescent labeled IN and performed single-molecule intensity and Förster resonance energy transfer experiments. The nuclear localization of the MLV pre-integration complex neither altered the IN content, nor its quaternary structure. Instead, BET-mediated interaction of the MLV intasome with chromatin in the post-mitotic nucleus reshaped its quaternary structure.

Authors : Borrenberghs Doortje, Zurnic Irena, De Wit Flore, Acke Aline, Dirix Lieve, Cereseto Anna, Debyser Zeger, Hendrix Jelle,

(6) IND-enabling studies for a clinical trial to genetically program a persistent cancer-targeted immune system.[TOP]

Pubmed ID :30409823
Publication Date : //
To improve persistence of adoptively transferred T cell receptor (TCR)-engineered T cells and durable clinical responses, we designed a clinical trial to transplant genetically-modified hematopoietic stem cells (HSCs) together with adoptive cell transfer (ACT) of T cells both engineered to express an NY-ESO-1 TCR. Here, we report the preclinical studies performed to enable an investigational new drug (IND) application.

Authors : Puig-Saus Cristina, Parisi Giulia, Garcia-Diaz Angel, Krystofinski Paige E, Sandoval Salemiz, Zhang Ruixue, Champhekar Ameya S, McCabe James, Cheung-Lau Gardenia C, Truong Nhat A, Vega-Crespo Agustin, Komenan Marie Desiles S, Pang Jia, Macabali Mignonette H, Saco Justin D, Goodwin Jeffrey L, Bolon Brad, Seet Christopher S, Montel-Hagen Amelie, Crooks Gay M, Hollis Roger P, Campo-Fernandez Beatriz, Bischof Daniela, Cornetta Kenneth, Gschweng Eric H, Adelson Celia, Nguyen Alexander, Yang Lili, Witte Owen N, Baltimore David, Coming-Anduix Begoña, Kohn Donald B, Wang Xiaoyan, Cabrera Paula, Kaplan-Lefko Paula J, Berent-Maoz Beata, Ribas Antoni,

(7) Phase 1 Trial of Autologous CAR T Cells Targeting NKG2D Ligands in Patients with AML/MDS and Multiple Myeloma.[TOP]

Pubmed ID :30396908
Publication Date : //
NKG2D ligands are widely expressed in solid and hematologic malignancies but absent or poorly expressed on healthy tissues. We conducted a phase 1 dose-escalation study to evaluate the safety and feasibility of a single infusion of NKG2D-chimeric antigen receptor (CAR) T cells, without lymphodepleting conditioning in subjects with acute myeloid leukemia/myelodysplastic syndrome or relapsed/refractory multiple myeloma. Autologous T cells were transfected with a γ-retroviral vector encoding a CAR fusing human NKG2D with the CD3ζ signaling domain. Four dose levels (1x10e6-3x10e7 total viable T cells) were evaluated. Twelve subjects were infused (7 AML, 5 MM). NKG2D-CAR products demonstrated a median 75% vector-driven NKG2D expression on CD3+ T cells. No dose-limiting toxicities, cytokine release syndrome, or CAR T cell-related neurotoxicity was observed. No significant autoimmune reactions were noted, and none of the >/= Grade 3 adverse events were attributable to NKG2D-CAR T cells. At the single injection of low cell doses employed in this trial, no objective tumor responses were observed. However, hematologic parameters transiently improved in one subject with AML at the highest dose, and cases of disease stability without further therapy or on subsequent treatments were noted. At 24 hours, the cytokine RANTES increased a median of 1.9-fold among all subjects and 5.8-fold among 6 AML patients. Consistent with preclinical studies, NKG2D-CAR T cell-expansion and persistence were limited. Manufactured NKG2D-CAR T cells exhibited functional activity against autologous tumor cells in vitro, but modifications to enhance CAR T-cell expansion and target density may be needed to boost clinical activity.

Authors : Baumeister Susanne H, Murad Joana, Werner Lillian, Daley Heather, Trebeden-Negre Helene, Gicobi Joanina K, Schmucker Adam, Reder Jake, Sentman Charles L, Gilham David Edward, Lehmann Frédéric F, Galinsky Ilene, DiPietro Heidi, Cummings Kristen, Munshi Nikhil C, Stone Richard M, Neuberg Donna S, Soiffer Robert, Dranoff Glenn, Ritz Jerome, Nikiforow Sarah,

(8) Dual-vector prodrug activator gene therapy using retroviral replicating vectors.[TOP]

Pubmed ID :30348946
Publication Date : //
Retroviral replicating vectors (RRVs) have been shown to achieve efficient tumor transduction and enhanced therapeutic benefits in a variety of cancer models. In the present study, we evaluated a possible combinatorial effect of prodrug activator genes delivered by two different RRVs derived from amphotropic murine leukemia virus (AMLV) and gibbon ape leukemia virus (GALV) on human hepatocellular carcinoma Hep3B cells. Both RRVs showed efficient replicative spread in culture and can overcame superinfection resistance each other. Notably, the replication and spread of each RRV in culture remained unaffected by pretransduction with the counterpart RRV. We further transduced cells with RRVs which individually possessed the prodrug activator genes yeast cytosine deaminase (CD) and herpes simplex virus thymidine kinase (TK) alone or in combination, and evaluated the cytotoxic effects of RRV-mediated gene therapy with CD and TK in the presence of the respective prodrugs, 5-fluorocytosine and ganciclovir. All combinations of the two prodrug activator genes produced synergistic cytocidal effects, but the combined effects of the different genes were significantly greater than those of the same genes when delivered by two different vectors. The present findings indicate the potential utility of dual-vector gene therapy using two different RRVs carrying different prodrug activator genes.

Authors : Kubo Shuji, Takagi-Kimura Misato, Tagawa Masatoshi, Kasahara Noriyuki,

(9) 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,

(10) 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,