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pBABEhygro_uPAR Retroviral Vector

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[#RTV-502] pBABEhygro_uPAR Retroviral Vector


RTV-502 | pBABEhygro_uPAR Retroviral Vector, 100 µL
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(1) Fluorescent genetic barcoding for cellular multiplex analyses.[TOP]

Pubmed ID :30098396
Publication Date : //
Hematopoiesis depends on the controlled differentiation of hematopoietic stem cells to mature cells with defined functions. Although each cell population within the hematopoietic hierarchy can be described by phenotypic markers, isolation of marker pure populations does not necessarily result in cells with homogeneous functionality. However, techniques that enable the efficient characterization of cell behavior with high resolution are limited. While single cell transplantation assays demand high mouse numbers and workload, sequencing-based fate tracking techniques require the destruction of the host cell, substantial financial resources, bioinformatics expertise, and suffer from a delay between sample acquisition and data interpretation. To make analyses more efficient, several laboratories recently developed flow cytometry-driven fluorescence-based multiplexing approaches that enable simultaneous parallel analysis of longitudinal behavior from multiple clonally-derived cells or polyclonal populations. Although these fluorescent genetic barcoding systems are still in their infancy, their power lies in the use of retroviral vectors for gene marking of multiple populations with unique fluorescent color codes. Tracing of color-coded cells by flow cytometry guarantees the accessibility of information on population behavior in real time and at low costs, supports the prospective isolation of cells for downstream analyses, and can be applied to cell line models as well as to human and animal-derived primary cells. This article discusses recent progress in the emerging field of fluorescent genetic barcoding for longitudinal multiplex cell tracking in biomedical research, and how this technique will help to uncover mechanisms regulating cell behavior with clonal resolution in a reduced number of experimental samples.

Authors : Maetzig Tobias, Morgan Michael, Schambach Axel,

(2) ALDH1A3 Is the Key Isoform That Contributes to Aldehyde Dehydrogenase Activity and Affects Proliferation in Cardiac Atrial Appendage Progenitor Cells.[TOP]

Pubmed ID :30087899
Publication Date : //
High aldehyde dehydrogenase (ALDH) activity has been reported in normal and cancer stem cells. We and others have shown previously that human ALDH cardiac atrial appendage cells are enriched with stem/progenitor cells. The role of ALDH in these cells is poorly understood but it may come down to the specific ALDH isoform(s) expressed. This study aimed to compare ALDH and ALDH atrial cells and to identify the isoform(s) that contribute to ALDH activity, and their functional role. Cells were isolated from atrial appendage specimens from patients with ischemic and/or valvular heart disease undergoing heart surgery. ALDH activity assessed with the Aldefluor reagent coincided with primitive surface marker expression (CD34). Depending on their ALDH activity, RT-PCR analysis of ALDH and ALDH cells demonstrated a differential pattern of pluripotency genes (Oct 4, Nanog) and genes for more established cardiac lineages (Nkx2.5, Tbx5, Mef2c, GATA4). ALDH cells, but not ALDH cells, formed clones and were culture-expanded. When cultured under cardiac differentiation conditions, ALDH cells gave rise to a higher number of cardiomyocytes compared with ALDH cells. Among 19 ALDH isoforms known in human, ALDH1A3 was most highly expressed in ALDH atrial cells. Knocking down ALDH1A3, but not ALDH1A1, ALDH1A2, ALDH2, ALDH4A1, or ALDH8A1 using siRNA decreased ALDH activity and cell proliferation in ALDH cells. Conversely, overexpressing ALDH1A3 with a retroviral vector increased proliferation in ALDH cells. ALDH1A3 is the key isoform responsible for ALDH activity in ALDH atrial appendage cells, which have a propensity to differentiate into cardiomyocytes. ALDH1A3 affects proliferation of these cells.

Authors : Puttini Stefania, Plaisance Isabelle, Barile Lucio, Cervio Elisabetta, Milano Giuseppina, Marcato Paola, Pedrazzini Thierry, Vassalli Giuseppe,

(3) Pharmacologically upregulated carcinoembryonic antigen-expression enhances the cytolytic activity of genetically-modified chimeric antigen receptor NK-92MI against colorectal cancer cells.[TOP]

Pubmed ID :30075754
Publication Date : //
The natural killer cell line, NK-92MI, is cytotoxic against various types of cancer. The aim of this study was to develop chimeric antigen receptor-modified (CAR) NK-92MI cells targeting carcinoembryonic antigen-expressing (CEA) tumours and increase killing efficacy by pharmacologically modifying CEA-expression.

Authors : Shiozawa Masayuki, Chang Chuan-Hsin, Huang Yi-Chun, Chen Yi-Ching, Chi Mau-Shin, Hao Hsu-Chao, Chang Yue-Cune, Takeda Satoru, Chi Kwan-Hwa, Wang Yu-Shan,

(4) Oncogenic heterogeneous nuclear ribonucleoprotein D-like promotes the growth of human colon cancer SW620 cells via its regulation of cell-cycle.[TOP]

Pubmed ID :30052712
Publication Date : //
Heterogeneous nuclear ribonucleoproteins (hnRNPs) represent a large family of RNA-binding proteins. Heterogeneous nuclear ribonucleoprotein D-like (HNRPDL) is a member of this family. Though aberrant expression of HNRPDL has been reported in a few cancers, whether HNRPDL is deregulated in colon cancer patients and what role this protein plays in these cells are not known yet. In this study, we found that HNRPDL was significantly up-regulated in colon cancer specimens than control. We also demonstrated that HNRPDL silencing inhibited the growth of SW620 cells both in vitro and in vivo. Conversely, we constructed a retroviral vector to deliver HNRPDL into non-malignant NIH-3T3 cells and injected these cells into nude mice. HNRPDL-overexpressing NIH-3T3 cells generated tumors in nude mice but not the control cells. Mechanistically, HNRPDL promoted cell-cycle progression associated with enhanced expressions of cyclin D3 and Ki-67 but decreased expressions of p53 and p21. Taken together, our data demonstrate that HNRPDL is aberrantly expressed in colon cancer cells, which promotes the growth of these cells by activating cell-cycle progression.

Authors : Zhang Pengshan, Ji Dehuan, Hu Xiaohui, Ni Hengli, Ma Wenjuan, Zhang Xiuyan, Liao Shibing, Zeng Zheng, Zhao Yun, Zhou Haixia,

(5) Efficient tumor transduction and antitumor efficacy in experimental human osteosarcoma using retroviral replicating vectors.[TOP]

Pubmed ID :30042500
Publication Date : //
Retroviral replicating vectors (RRVs) have achieved efficient tumor transduction and enhanced therapeutic benefit in a wide variety of cancer models. Here, we evaluated two different RRVs derived from amphotropic murine leukemia virus (AMLV) and gibbon ape leukemia virus (GALV), which utilize different cellular receptors (PiT-2 and PiT-1, respectively) for viral entry, in human osteosarcoma cells. Quantitative RT-PCR showed that low levels of expression of both receptors were observed in normal and non-malignant cells. However, high PiT-2 (for AMLV) and low PiT-1 (for GALV) expression was observed in most osteosarcoma cell lines. Accordingly, AMLV expressing the green fluorescent protein gene infected and replicated more efficiently than GALV in most osteosarcoma cell lines. Furthermore, RRVs expressing the cytosine deaminase prodrug activator gene showed differential cytotoxicity that correlated with the results of viral spread. AMLV-RRV-mediated prodrug activator gene therapy achieved significant inhibition of subcutaneous MG-63 tumor growth over GALV in nude mice. These data indicate that AMLV vectors predominate over GALV in human osteosarcoma cells. Moreover, our findings support the potential utility of the two RRVs in personalized cancer virotherapy on the basis of receptor expression.

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

(6) Bacteria-free minicircle DNA system to generate integration-free CAR-T cells.[TOP]

Pubmed ID :30030293
Publication Date : //
Chimeric antigen receptor T (CAR-T) cells engineered with lentiviral and retroviral vectors have been successfully applied to treat patients with B cell malignancy. However, viral integration in T cells has the potential risk of mutagenesis, and viral vector production demands effort and is costly. Using non-integrative episomal vector such as minicircle vector to generate integration-free CAR-T cells is an attractive option.

Authors : Cheng Chen, Tang Na, Li Jiaxin, Cao Shiwei, Zhang Tongtong, Wei Xiaofei, Wang Haoyi,

(7) Influence of retronectin-mediated T cell activation on expansion and phenotype of CD19-specific chimeric antigen receptor T cells.[TOP]

Pubmed ID :30024314
Publication Date : //
Introduction Enhanced in vivo expansion, long-term persistence of chimeric antigen receptor T (CART) cells and efficient tumor eradication through these cells are linked to the proportion of less-differentiated cells in the CART cell product. Retronectin is well established as an adjuvant for improved retroviral transduction while its property to enrich less-differentiated T cells is less known. In order to increase these subsets, we investigated effects of retronectin-mediated T cell activation for CD19-specific CART cell production. Materials and Methods Peripheral blood mononuclear cells (PBMCs) of healthy donors (HDs) and untreated chronic lymphocytic leukemia (CLL) patients without or with positive selection for CD3+ T cells were transduced with a CD19.CAR.CD28.CD137zeta 3rd generation retroviral vector. Activation of PBMCs was performed by CD3/CD28, CD3/CD28/retronectin or CD3/retronectin. Interleukin (IL)-7 and IL-15 were supplemented to all cultures. Retronectin was used in all three activation protocols for retroviral transduction. Expansion was assessed by trypan blue staining. Viability, transduction efficiency, immune phenotype and cytokine production were longitudinally analyzed by flow cytometry. Cytotoxic capacity of generated CART cells was evaluated using a classical chromium-51 release assay. Results Retronectin-mediated activation resulted in an enrichment of CD8+ cytotoxic CART cells and less-differentiated naïve-like T cells (CD45RA+CCR7+). Retronectin-activated CART cells showed increased cytotoxic activity. However, activation with retronectin decreased viability, expansion, transduction efficiency and cytokine production, particularly of CLL patient-derived CART cells. Conclusion Both retronectin-mediated activation protocols promoted a less-differentiated CART cell phenotype without comprising cytotoxic properties of HD-derived CART cells. However, up-front retronectin resulted in reduced viability and expansion in CLL patients. This effect is probably attributed to the retronectin-mediated activation of B cells with prolonged CLL-persistence. Consequently, CART cell expansion and generation failed. In summary, activation with retronectin should be performed with caution and may be limited to patients without a higher percentage of tumor cells in the peripheral blood.

Authors : Stock Sophia, Hoffmann Jean-Marc, Schubert Maria-Luisa, Wang Lei, Wang Sanmei, Gong Wenjie, Neuber Brigitte, Gern Ulrike, Schmitt Anita, Müller-Tidow Carsten, Dreger Peter, Schmitt Michael, Sellner Leopold,

(8) Amino acid deprivation triggers a novel GCN2-independent response leading to the transcriptional reactivation of non-native DNA sequences.[TOP]

Pubmed ID :30020994
Publication Date : //
In a variety of species, reduced food intake, and in particular protein or amino acid (AA) restriction, extends lifespan and healthspan. However, the underlying epigenetic and/or transcriptional mechanisms are largely unknown, and dissection of specific pathways in cultured cells may contribute to filling this gap. We have previously shown that, in mammalian cells, deprivation of essential AAs (methionine/cysteine or tyrosine) leads to the transcriptional reactivation of integrated silenced transgenes, including plasmid and retroviral vectors and latent HIV-1 provirus, by a process involving epigenetic chromatic remodeling and histone acetylation. Here we show that the deprivation of methionine/cysteine also leads to the transcriptional upregulation of endogenous retroviruses, suggesting that essential AA starvation affects the expression not only of exogenous non-native DNA sequences, but also of endogenous anciently-integrated and silenced parasitic elements of the genome. Moreover, we show that the transgene reactivation response is highly conserved in different mammalian cell types, and it is reproducible with deprivation of most essential AAs. The General Control Non-derepressible 2 (GCN2) kinase and the downstream integrated stress response represent the best candidates mediating this process; however, by pharmacological approaches, RNA interference and genomic editing, we demonstrate that they are not implicated. Instead, the response requires MEK/ERK and/or JNK activity and is reproduced by ribosomal inhibitors, suggesting that it is triggered by a novel nutrient-sensing and signaling pathway, initiated by translational block at the ribosome, and independent of mTOR and GCN2. Overall, these findings point to a general transcriptional response to essential AA deprivation, which affects the expression of non-native genomic sequences, with relevant implications for the epigenetic/transcriptional effects of AA restriction in health and disease.

Authors : De Vito Annarosaria, Lazzaro Massimo, Palmisano Ilaria, Cittaro Davide, Riba Michela, Lazarevic Dejan, Bannai Makoto, Gabellini Davide, Schiaffino Maria Vittoria,

(9) Physical characterization and stabilization of a lentiviral vector against adsorption and freeze-thaw.[TOP]

Pubmed ID :30017889
Publication Date : //
A replication-deficient lentiviral vector encoding the tumor antigen gene NY-ESO-1 was characterized in terms of vector morphology, particle size range, concentration and zeta potential employing a variety of physical methods. Environmentally stressed vector samples were then evaluated in terms of viral vector particle size and concentration by nanoparticle tracking analysis (NTA). These NTA stability results correlated reasonably well with a qPCR assay for quantitation of viral genome copy number (r2=0.80). Approximately 40 pharmaceutical excipients were examined for their ability to stabilize the vector against exposure to an adsorptive container surface (glass) as well as freeze-thaw cycling using NTA as the screening method. Stabilizing additives that inhibit viral vector particle loss under these conditions included proline, lactose and mannitol. Several candidate frozen liquid formulations that contained a combination of these lead excipients and various buffering agents were further evaluated for their ability to stabilize the viral vector. The additional benefit of lowering the Tris buffer concentration was observed. This study highlights the use of physical particle assays such as NTA for initial screening of stabilizing excipients to minimize vector loss due to container adsorption and freeze-thaw cycling to facilitate early formulation development of viral vector candidates in frozen liquid formulations.

Authors : Kumru Ozan S, Wang Yu, Gombotz C Wayne R, Kelley-Clarke Brenna, Cieplak Witold, Kim Tae, Joshi Sangeeta B, Volkin David B,

(10) Continuous production process of retroviral vector for adoptive T- cell therapy.[TOP]

Pubmed ID :29977134
Publication Date : //
Adoptive T-Cell therapy is being considered as a promising method for cancer treatment. In this approach, patient's T cells are isolated, modified, expanded, and administered back to the patient. Modifications may include adding specific T cell receptors (TCR) or chimeric antigen receptors (CAR) to the isolated cells by using retroviral vectors. PG13 cells, derivatives of NIH3T3 mouse fibroblasts, are being used to stably produce retroviral vectors that transduce the T cells. PG13 cells are anchorage-dependent cells that grow in roller bottles or cell factories and lately also in fixed bed bioreactors to produce the needed viral vector. To scale up viral vector production, PG13 cells were propagated on microcarriers in a stirred tank bioreactor utilizing an alternating tangential flow perfusion system. Microcarriers are 10 µm - 0.5 mm beads that support the attachment of cells and are suspended in the bioreactor that provides controlled growth conditions. As a result, growth parameters, such as dissolved oxygen concentration, pH, and nutrients are monitored and continuously controlled. There were no detrimental effects on the specific viral vector titer or on the efficacy of the vector in transducing the T cells of several patients. Viral vector titer increased throughout the 11 days perfusion period, a total of 4.8 × 10 transducing units (TU) were obtained with an average titer of 4.4 × 10 TU/mL and average specific productivity of 10.3 (TU) per cell, suggesting that this method can be an efficient way to produce large quantities of active vector suitable for clinical use.

Authors : Inwood Sarah, Xu Hui, Black Mary A, Betenbaugh Michael J, Feldman Steven, Shiloach Joseph,