Recombinant Retroviral Vector Research Articles

ELF4 promotes proliferation and inhibits apoptosis of human insulinoma cells by activating Akt signaling

To investigate the effect of overexpression of the oncogenic transcription factor ELF4 on proliferation and apoptosis in human insulinoma cells and explore the underlying mechanism. A human insulinoma BON cell line with stable overexpression of ELF4 (BON-ELF4 cells) was constructed using a recombinant retrovirus vector and the expression of ELF4 protein was verified using Western blotting. MTT assay was used to assess the proliferation of BON-ELF4 cells and BON-Vector cells, and the cell apoptosis induced by treatment with epirubicin (0.1 μmol/L for 24 h) was analyzed by detecting the expressions of cleaved caspase-8, caspase-9, and PARP using Western blotting. Flow cytometry with Annexin VFITC/PI staining was performed to analyze the numbers of apoptotic BON-Vector or BON-ELF4 cells. The expressions of phosphorylated Akt and total Akt in the cells were detected using Western blotting. BON-ELF4 cell line with stable overexpression of ELF4 was successfully established. ELF4 overexpression significantly promoted the proliferation (P < 0.05) and obviously suppressed epirubicin- induced apoptosis in BON cells, resulting also in significantly reduced expressions of cleaved caspase-8, caspase-9 and PARP (P < 0.05). The results of flow cytometry showed a significantly lower apoptotic rate in BON-ELF4 cells than in BON-Vector cells following epirubicin treatment (6.03% vs 22.90%). The phosphorylation levels of Akt (Thr308 and Ser473) were significantly increased (P < 0.05) while the level of total Akt remained unchanged (P>0.05) in ELF4- overexpressing cells. ELF4 overexpression enhances the proliferation and suppresses apoptosis of insulinomas cells by activating Akt signaling.

Establishing cGMP manufacturing of CRISPR/Cas9-edited human CAR T cells

Background & Aim Chimeric antigen receptors (CARs) are synthetic receptors that redirect and reprogram T cells to mediate tumor rejection. CD19 targeted CAR T cells have showed remarkable effacy for chemorefractory/relapsed B cell malignancies. Recombinant retroviral vectors mediated- CAR gene transfer is currently the standard method to generate cGMP grade CAR T cells. However, CAR gene expression is highly variable, owing to position effects and vector incorporation variations. CAR T cells engineered in this manner are capable of tumor eradication, but are prone to tonic signaling and accelerated exhaustion. We have developed a novel genetic engineering strategy to insert CAR genes into a precise genomic location in human peripheral blood T cells. Methods, Results & Conclusion T cells are electroporated with Cas9 mRNA or Cas9 protein and a guide RNA, followed by transduction with a recombinant Adeno-Associated Virus encoding the CAR sequence to facilitate the insertion of the CAR gene upstream of the constant region of TCR alpha chain. This results in the endogenous TCR promoter (TRAC) controlled CAR expression and abrogation of TCR surface expression. This strategy not only allows uniform CAR expression, but also delays T-cell differentiation and exhaustion, leading to enhanced T cell function and anti-tumor efficacy. The edited cells vastly outperformes retrovirally modified CAR T cells in a pre-B ALL NALM6 mouse model. The targeting of CARs to the TCR locus also provides a safer therapeutic T cell by minimizing the risks of insertional oncogenesis, and TCR-induced autoimmunity and alloreactivity (thus spanning both autologous and allogeneic T cell applications). In addition, we have incorporated in the CAR a mutant CD3z chain encoding a single immunoreceptor tyrosine-based activation motif that improves the balance between effector and memory T cells composition. We are in the process of translating this novel approach into the clinical setting by establishing cGMP conditions and protocols for the manufacturing of TRAC-CAR T cells. Using the 4D-LV electroporator, we have demonstrated that we can knockout the TCR at large scale (100 E06 CD3+ T cells) with high efficiency (70-80%) as that obtained at small scale. We have evaluated AAV6 to deliver the CAR transgene in the TCR locus. Data will be presented on optimizing the manufacturing of the TRAC-CAR T cells and on evaluating their anti-tumor efficacy in vivo.

Abstract A69: Identification of HLA-A0201 restricted epitope of cancer/testis antigen (CTA) Hormad1 and generation of antigen-specific T-cell receptor-engineered T cells (TCR-T) for treatment of solid tumor malignancies

Abstract Purpose: Hormad1 is a novel cancer/testis antigen (CTA) that is highly expressed in several solid tumor types such as lung cancer, esophageal carcinoma, as well as head and neck cancer. High expression of Hormad1 is correlated with elevated mutational burden and reduced survival in lung adenocarcinoma, indicating that Hormad1 could be a potential target for immunotherapy. The aims of this study are to identify HLA-A0201-restricted epitope of Hormad1 and generate antigen-specific T cells and T-cell receptor (TCR)-engineered T cells (TCR-T) for adoptive cell therapy (ACT) to treat patients with Hormad1-expressing solid tumors. Methods: Candidate HLA-A0201 restricted epitopes of Hormad1 were identified by reverse-immunology and MHC class I stabilization assay. Mature HLA-A0201+ dendritic cells (mDC) were pulsed with the candidate epitope peptides and used to stimulate autologous CD25 depleted T cells. Tetramer guided sorting was used to enrich for Hormad1-specific T cells from which CTL cell lines and clones were generated. The alpha and beta TCR chains were sequenced from HORMAD1-specific, tumor-reactive CTL and cloned into a retroviral vector construct to introduce the TCR to allogeneic PBMC to generate functional TCR-T cells. Results: From 6 candidate peptides, peptide 4, which showed highest HLA-A2 binding characters, was chosen for T-cell generation. After stimulation with peptide pulsed mDC, functional CTL cell line and clones were generated from two healthy donor PBMC. Both the CTL cell line and clone selectively recognize tumor targets expressing HLA-A0201 and Hormad1. Specificity of the CTL for the HORMAD1 epitope presented by HLA-A0201 was confirmed using lines coexpressing HLA-A0201 and/or HORMAD1. Cold target inhibition assays further confirmed that the Hormad1 peptide 4 was naturally processed and presented by tumor targets. After transduction with the recombinant retroviral vector expressing the alpha and beta TCR chains of the functional CTL cell line, the resultant TCR-T cells displayed high affinity, specific tumor target killing at E:T of 1: 1.25. Moreover, these TCR-T cells specifically enhanced CD137, CD69, IFN-γ, and TNF-α expression in response to HLA-A0201-positive and Hormad1-positive tumor target cells. Conclusion: The HLA-A0201 restricted Hormad1 epitope identified in this study is a novel candidate peptide for antigen-specific vaccine development and endogenous T-cell therapy. A Hormad1-specific TCR-encoding vector was also created that conferred tumor reactivity and antigen specificity among TCR-T cells, offering a high-value product for use in adoptive T-cell therapy for patients with Hormad1-expressing solid tumor malignancies. Citation Format: Ke Pan, Yulun Chiu, Farah Hasan, Natalia Miroballi, Angelique Whitehurst, Cassian Yee. Identification of HLA-A0201 restricted epitope of cancer/testis antigen (CTA) Hormad1 and generation of antigen-specific T-cell receptor-engineered T cells (TCR-T) for treatment of solid tumor malignancies [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A69.

The implication of Numb-induced Notch signaling in endothelial-mesenchymal transition of diabetic nephropathy

AimThis study was purposed to figure out the contribution of Numb-induced Notch signaling to the development of diabetic nephropathy (DN). MethodsTwo hundred and twenty six DN patients were included, and human glomerular endothelial cells (RGEC) were cultured. MSCV-Numb-IRES-GFP, MSCV-Notch1-IRES-GFP and MSCV-Hes1-IRES-GFP were transfected to construct the recombinant retroviral vectors. ResultThe over-expressed Numb and Notch1, as well as the under-expressed Hes-1 were correlated with the undesirable prognosis of DN patients (P < 0.05). Within the cell lines transfection with si-Numb would cut down E-cadherin and CD31 expressions (P < 0.05), yet elevated α-SMA and vimentin expressions (P < 0.05). The apoptotic rate of si-Numb cell lines underperformed ones categorized into the hyperglucose group (P < 0.05), whereas the lowly-expressed Notch1 and Hes1 were observably associated with inhibited proliferation of myofibroblasts (P < 0.05). Addition of ADPT caused under-expressed α-SMA and vimentin, along with the over-expressed E-cadherin and CD31 (P < 0.05). Silencing of Notch1 and Hes1 reversed the epithelial-mesenchymal transition (EMT) process that was triggered by high glucose (P < 0.05). ConclusionNumb negatively regulated Notch signaling pathway in EMT of DN, implying that they had great potentials to serve as therapeutic targets or diagnostic biomarkers for DN.

Expression of Exogenous Genes in Murine Primary B Cells and B Cell Lines Using Retroviral Vectors.

B cells, after activation, can undergo class-switch recombination and somatic hypermutation of their immunoglobulin genes, and can differentiate into memory cells and plasma cells. Expressing genes in altered versions in primary B cells and B cell lines is an important approach to understanding how B cell receptor signaling leads to B cell activation and differentiation. Recombinant retrovirus-based transduction is the most efficient method to deliver exogenous genes for expression in B cells. In this chapter, we describe streamlined protocols for using recombinant retroviral vectors to transduce both murine primary B cells and B cell lines.

Preparation of recombinant retrovirus pRevTRE-E77.43 and its protective effect in a mouse model of Schistosoma japonicum infection

To explore the biological functions of E77.43, a gene segment of Microtus fortis, in treating Schistosoma japonicum infection. Recombinant retroviral vectors of pRevTRE-E77.43 was constructed, and recombinant retroviral vectors were transfected into PA317 cells, and the stable cell lines were obtained by hygromycin screening, followed by the packaging, concentration and purification of recombinant retrovirus. The virus was transferred to the mice infected by S. japonicum via intravenous or intraperitoneal injection, through which the express of target gene and the treatment function in vivo were observed. The experiment showed the recombinant virus injected mice could efficiently express E77.43 on the 7th day after the injection which lasted for forty-five days thereafter. A significant reduction in adult worms (31.0%) and a high reduction (35.0%) in liver eggs were induced by pRevTRE-E77.43, while the reduction in adult worms and that in liver eggs was 1.2% and 0.9% induced by pRevTRE respectively (t = 3.524, 9.485, both P＜0.01). pRevTRE-E77.43 could be used for the treatment of S. japonicum infection, indicating that E77.43 may involve in the natural resistance of M. fortis to S. japonicum infection.

Inhibitory effect of RNA interference targeting human telomerase reverse transcriptase against hepatoblastoma cells

Objective To construct a recombinant retrovirus vector expressing small interfering RNA (siRNA) targeting human telomerase reverse transcriptase (hTERT), and assess its effect on proliferation and apoptosis of human hepatoblastoma cells. Methods The sequences of the siRNA targeting hTERT, U6 promoter and enhanced green fluorescent protein (EGFP) gene were amplified by polymerase chain reaction (PCR) and inserted into the mammalian retroviral expression vector pLXSN. The PCR method was used to amplify hTERT-siRNA, EGFP, DNA fragment of U6+ 27. The retroviral expression vector pLXSN-EGFP-U6-siTERT was constructed and subjected to enzyme digestion identification. The recombinant retroviral vector pLXSN-EGFP-U6-siTERT was constructed. The vector was then used to infect human hepatoblastoma cell HepG2. The telomerase activity of the infected cells was detected by telomerase repeat amplification protocol-silver staining, and the cell apoptosis was examined using flow cytometry. The inhibition rate of HepG2 cell proliferation was analyzed by methyl thiazol tetrazolium (MTT) assay. Results hTERT-siRNA retroviral expression vectors were successfully prepared. In the control group, the telomerase activity was 2 143.06±198.69. At 24, 48 and 72 h after colony forming units (CFU) recombinant virus infection the telomerase activity was 1 632.02±116.28, 899.38±126.11 and 321.25±25.25 respectively. As compared with before infection, the telomerase activity was respectively decreased by 23.84%, 58.03% and 85.01%. There was significant difference among different time groups (P=0.046, 0.024 and 0.008). The results of flow cytometry showed that there was significant difference in the cell apoptosis rate between the experimental group and the control group with different titer of recombinant virus infection, and there was a concentration-dependent relationship. At 24 h after 1×105 CFU recombinant virus infection, the apoptosis rate was 29.05%. MTT results showed that apoptosis rate was 29.05% at 24 h after recombinant virus infection, the tumor cell death was significant, and with the increase in virus titer and prolongation of time, cell death also gradually increased. At 24, 48 and 72 h after virus infection of 6.0×105 CFU, the results of MTT assay were 0.29±0.14, 0.20±0.13 and 0.18±0.11 respectively. At 24, 48 and 72 h after virus infection of 3.0×105 CFU, MTT results were 0.32±0.11, 0.26±0.12 and 0.25±0.10. At 24, 48 and 72 h after virus infection of 1.0×105 CFU, results of MTT assay were 0.33±0.12, 0.26±0.13 and 0.26±0.12. At 24, 48 and 72 h after virus infection of 1.0×104 CFU, the results of MTT assay were 0.43±0.14, 0.35±0.10 and 0.33±0.15. At 24, 48 and 72 h after virus infection of 1.0×103 CFU, the results of MTT assay were 0.52±0.11, 0.44±0.13 and 0.44±0.10. At 24, 48 and 72 h after virus infection of 1.0×102 CFU, the results of MTT assay were 0.65±0.13, 0.61±0.15 and 0.60±0.16. In the negative control group, the results of MTT assay were 0.69±0.11, 1.01±0.14 and 2.98±0.16 respectively at 24, 48 and 72 h. There existed the concentration-effect and time-effect relationship (P=0.037, 0.034 and 0.028). Conclusion hTERT-siRNA can effectively silence hTERT gene and suppress the telomerase activity and proliferation of HepG2 cells. Key words: RNA interference; Human telomerase reverse transcriptase; Hepatoblastoma; Apoptosis

Establishment and Characterization of an SV40 Large T Antigen-Transduced Porcine Colonic Epithelial Cell Line

Continuous cell lines have become indispensable tools that have enabled investigations into cellular mechanisms by increasing experimental reproducibility and sample availability, and decreasing the use of experimental animals. To facilitate studies of epithelial barrier function of the porcine colon, we aimed to establish an epithelial cell line with an extended replicative capacity. Cells were isolated from the proximal colon of a 3-week-old piglet and transduced using a recombinant retroviral vector construct containing the simian virus 40 large T antigen (SV40 TAg). We established a clonal epithelial cell line, referred to as PoCo83-3, that stably expressed the SV40 TAg, verified at mRNA and protein levels. PoCo83-3 showed epithelial cell-specific features, such as cobblestone-like morphology, dome structure formation, the presence of apical microvilli, and the expression of keratin 18, E-cadherin and the tight junction-associated proteins zonula occludens-1, occludin, and claudin-1. To validate PoCo83-3 as an in vitro model in epithelial barrier research, proinflammatory cytokine-inducible alterations in barrier integrity were demonstrated by incubating the cells with TNF-α and IFN-γ for 48 h. These cytokine treatments promoted a decreased transepithelial electrical resistance. In summary, PoCo83-3 exhibited an extended life span and a differentiated phenotype while maintaining epithelial characteristics. Based on these results, we present this cell line as a valuable in vitro model for investigations of epithelial barrier function in the porcine colon.

A rapid and efficient polyethylenimine-based transfection method to prepare lentiviral or retroviral vectors: useful for making iPS cells and transduction of primary cells.

To improve the efficiency, reproducibility and consistency of the PEI-based transfection method that is often used in preparation of recombinant lentiviral or retroviral vectors. The contributions to transfection efficiency of multi-factors including concentration of PEI or DNA, dilution buffer for PEI/DNA, manner to prepare PEI/DNA complexes, influence of serum, incubation time for PEI/DNA complexes, and transfection time were studied. Gentle mixing during the preparation of PEI/DNA transfection complexes is critical for a high transfection efficiency. PEI could be stored at room temperature or 4°C, and most importantly, multigelation should be avoided. The transfection efficiency of the PEI-based new method in different types of cells, such as 293T, Cos-7, HeLa, HepG2, Hep3B, Huh7 and L02, was also higher than that of the previous method. After optimization, the titer of our lentiviral system or retroviral system produced by PEI-based new method was about 10- or 3-times greater than that produced by PEI-based previous method, respectively. We provide a rapid and efficient PEI-based method for preparation of recombinant lentiviral or retroviral vectors which is useful for making iPS cells as well as transduction of primary cell cultures.

400. CD269 (BCMA)-Specific CAR-Expressing T Cells Dramatically Eradicate Myeloma Cells from Bone Marrow of an Orthotopic Multiple Myeloma Mouse Model

Introduction: Multiple myeloma (MM) is an incurable hematological malignancy of plasma cells. During the past decade, overall survival rate of multiple myeloma have been improved. These improvements are linked to the induction of novel drugs with different mechanism of action such as proteasome inhibitors and immnomodulatory drugs. However, MM remains in most cases an incurable disease, and new therapeutic strategies are urgently required for radical cure or continued disease control. During recent years, some dramatic responses were reported using T cells expressing CD19-targeted chimeric antigen receptors (CAR). Therefore, CAR therapy could also be a promising new therapeutic strategy for MM. CD269 (also known as BCMA: B-cell maturation antigen) is a membrane protein that is selectively expressed on B-cell lineages and on plasma cells including myeloma cells. Our goal of this study is to verify whether treatment with CD269-specific CAR-expressing T cells can eradicate myeloma cells from bone marrow of tumor-bearing NOG mice. Methods: Firstly, we uniquely developed monoclonal antibodies against human CD269. Next, we designed and verified novel CD269-specific CAR. The CD269-CAR recombinant retroviral vector encodes the MoMLV (Moloney murine leukemia virus) retroviral backbone and the 3.3E-28z CAR. The 3.3E-28z CAR consists of an anti-human CD269 scFv that was derived from the 3.3E mouse hybridoma, a portion of the human CD28 molecule and the intracellular domain of the human CD3ζ molecule. Results: CD269-specific CAR-expressing T cells show redirected cytolysis toward CD269-positive U266 human MM-derived cells, but not CD269-negative K562 cells. Luciferase-expressing U266 cells that were injected into the cardiac chamber of NOG mice, selectively infiltrated to bone marrow. Six weeks after tumor inoculation, we injected saline, non-CAR gene-modified T cells, or CAR gene-modified T cells (GMCs) into the cardiac chamber of tumor-bearing mice. In the group of GMC injection, U266 cells were dramatically eradicated from bone marrow. Conclusion: Although the efficacy of BCMA-CAR has been demonstrated by previous study using intradermal models of MM, our challenge is the first report that presents with orthotopic models of MM. Our results are more appropriate for predicting the efficacy of CD269-CAR in the treatment of MM. We conclude that adoptive transfer of CD269-CAR-expressing T cells could be a promising option for patient with MM.

Human CD8(+) T cells transduced with an additional receptor bispecific for both Mycobacterium tuberculosis and HIV-1 recognize both epitopes.

Tuberculosis (TB) and human immunodeficiency virus type 1 (HIV‐1) infection are closely intertwined, with one‐quarter of TB/HIV coinfected deaths among people died of TB. Effector CD8+ T cells play a crucial role in the control of Mycobacterium tuberculosis (MTB) and HIV‐1 infection in coinfected patients. Adoptive transfer of a multitude of effector CD8+ T cells is an appealing strategy to impose improved anti‐MTB/HIV‐1 activity onto coinfected individuals. Due to extensive existence of heterologous immunity, that is, T cells cross‐reactive with peptides encoded by related or even very dissimilar pathogens, it is reasonable to find a single T cell receptor (TCR) recognizing both MTB and HIV‐1 antigenic peptides. In this study, a single TCR specific for both MTB Ag85B199‐207 peptide and HIV‐1 Env120‐128 peptide was screened out from peripheral blood mononuclear cells of a HLA‐A*0201+ healthy individual using complementarity determining region 3 spectratype analysis and transferred to primary CD8+ T cells using a recombinant retroviral vector. The bispecificity of the TCR gene‐modified CD8+ T cells was demonstrated by elevated secretion of interferon‐γ, tumour necrosis factor‐α, granzyme B and specific cytolytic activity after antigen presentation of either Ag85B199‐207 or Env120‐128 by autologous dendritic cells. To the best of our knowledge, this study is the first report proposing to produce responses against two dissimilar antigenic peptides of MTB and HIV‐1 simultaneously by transfecting CD8+ T cells with a single TCR. Taken together, T cells transduced with the additional bispecific TCR might be a useful strategy in immunotherapy for MTB/HIV‐1 coinfected individuals.

Inhibition of human telomerase reverse transcriptase in vivo and in vitro for retroviral vector-based antisense oligonucleotide therapy in ovarian cancer.

Human telomerase is absent in most normal tissues, but is abnormally activated in all major cancer cells. Telomerase enables tumor cells to maintain telomere length, allowing indefinite replicative capacity. Albeit not sufficient in itself to induce neoplasia, telomerase is believed to be necessary for cancer cells to grow without limit. Studies using an antisense oligonucleotide (ASODN) to the RNA component of telomerase or human telomerase reverse transcriptase (hTERT) demonstrate that telomerase in human tumor lines can be blocked in vivo. Inhibition of hTERT led to telomere shortening and cancer cell death, validating telomerase as a target for anticancer genetic therapy. Varieties of approaches for hTERT inhibition have been investigated. The aim of this study was to analyze the biological activity of ASODN to the hTERT mediated by retrovirus vector, which was used as therapy for ovarian tumor. We constructed and characterized a recombinant retrovirus vector with full-length hTERT antisense complementary DNA. The vector was introduced into ES-2 by lipofectamine-mediated gene transfection. The cellular proliferation and telomerase activity of the transformant cells were retarded. The hTERT gene expression and the telomerase activity of the transformant cells were both decreased. The transformant cells show partial reversion of the malignant phenotype. PT67 cells were also transfected with the recombinant vector and virus-producer cells were generated. The retrovirus-containing supernatant effectively inhibited the growth of human ovarian tumor xenografts in mouse models (subcutaneous tumor model), and enhanced the mouse survival time.

Increased titer and reduced lactate accumulation in recombinant retrovirus production through the down-regulation of HIF1 and PDK.

Many mammalian cell lines used in the manufacturing of biopharmaceuticals exhibit high glycolytic flux predominantly channeled to the production of lactate. The accumulation of lactate in culture reduces cell viability and may also decrease product quality. In this work, we engineered a HEK 293 derived cell line producing a recombinant gene therapy retroviral vector, by down-regulating hypoxia inducible factor 1 (HIF1) and pyruvate dehydrogenase kinase (PDK). Specific productivity of infectious viral titers could be increased more than 20-fold for single gene knock-down (HIF1 or PDK) and more than 30-fold under combined down-regulation. Lactate production was reduced up to 4-fold. However, the reduction in lactate production, alone, was not sufficient to enhance the titer: high-titer clones also showed significant enrollment of metabolic routes not related to lactate production. Transcriptome analysis indicated activation of biological amines metabolism, detoxification routes, including glutathione metabolism, pentose phosphate pathway, glycogen biosynthesis and amino acid catabolism. The latter were validated by enzyme activity assays and metabolite profiling, respectively. High-titer clones also presented substantially increased transcript levels of the viral genes expression cassettes. The results herein presented demonstrate the impact of HIF1 and PDK down-regulation on the production performance of a mammalian cell line, reporting one of the highest fold-increase in specific productivity of infectious virus titers achieved by metabolic engineering. They additionally highlight the contribution of secondary pathways, beyond those related to lactate production, that can be also explored to pursue improved metabolic status favoring a high-producing phenotype.

Effect of Knockdown of Vascular Cell Adhesion Molecule-1 on the Immunologic Regulation Capacity of Murine Mesenchymal Stem Cells

To establish the stably lower expression of vascular cell adhesion molecule-1 (VCAM-1) in MSC cell line (C3H10T1/2) by siRNA technology, and explore the effect of knockdown of VCAM-1 on the immunologic regulation capacity of murine MSC. The mouse GV118-VCAM-1-RNAi retrovirus vector was constructed by gene recombination technology. The recombinant plasmid was identified by restriction analysis and sequencing, and then the recombinant plasmid GV118-VCAM-1-RNAi was transfected into 293 cells by Lipofectamine, and the supernatant was collected to transfect C3H10T1/2. Moreover, the VCAM-1 lower expression on MSC was evaluated by flow cytometry and fluorescent microscopy. The knockdown VCAM-1 MSC was sorted by flow cytometry. Furthermore, the inhibitory effect of the knockdown VCAM-1 MSC on lymphocyte proliferation was tested by lymphoblast transformation assay (LTT) and mixed lymphocyte reaction assay(MLR). The recombinant retroviral vector of knockdown VCAM-1 (GV118-VCAM-1-RNAi) was successfully constructed and transfected into mouse MSC cell line C3H10T1/2. The knockdown VCAM-1/MSC was obtained by flow cytometric sorting. The LTT and MLR assay showed that the immunosuppressive effect of MSC lower-expressing VCAM-1 dramatically decreased (P<0.05). Knockdown VCAM-1 in MSC can significantly down-regulate the inhibitory capability of MSC on the proliferation of T-cells. The data of this study laid an experimental foundation for studying effect of VCAM-1 transfecting into MSC on immune function.

Construction and identification of human glial cell-derived neurotrophic factor gene-modified Schwann cells from rhesus monkeys.

The objective of this study was to construct stable rhesus monkey Schwann cells (SCs) modified with the human glial cell-derived neurotrophic factor (hGDNF) gene. hGDNF gene amplification was performed with pUC19-hGDNF as templates, and then the coding sequence of hGDNF was inserted into the eukaryotic expression vector pBABE-puro to obtain the recombinant vector pBABE-puro-hGDNF. The recombinant vector pBABE-puro-hGDNF was identified with restriction enzyme, and then underwent DNA sequencing. SCs from rhesus monkeys were transfected with the recombinant vector pBABE-puro-hGDNF, and then the expression levels of mRNA and protein of the hGDNF gene were determined with real-time fluorescence quantitative PCR and Western blot, respectively, in the transfected SCs. The biological activity of GDNF gene-modified SCs (GDNF-SCs) was assessed by MTT assay. The length of the hGDNF coding sequence of PCR products was 569 bp. After the recombinant eukaryotic expression vectors were digested with restriction enzyme, there was a specific segment of 596 bp. The results of DNA sequencing of the specific segment of 596 bp were the same as that of hGDNF in GenBank, suggesting that the hGDNF gene was successfully inserted into the recombinant retrovirus vectors. The expression levels of mRNA and protein were significantly higher in transfected SCs as compared to nontransfected SCs (p<0.05). MTT assay indicated that the OD value was significantly higher in GDNF-SCs group than in SCs and DMEM groups (p<0.05). hGDNF-SCs can steadily and efficiently release hGDNF. This study provides a basis for cell therapy of nerve injury.

Overexpression of the PLAP-1 gene inhibits the differentiation of BMSCs into osteoblast-like cells.

Periodontal ligament-associated protein-1 (PLAP-1) is a newly discovered member of the extracellular matrix family of proteins known as proteoglycans and is a negative regulator that plays a crucial role in the homeostasis of periodontal tissues. It can protect the periodontal ligament from excessive osteogenesis. However, the molecular mechanisms of PLAP-1 during osteogenic differentiation and osteogenesis remain unclear. In this study, we constructed a PLAP-1 recombinant retroviral plasmid vector named pBABE-hygro-PLAP-1. We transfected this plasmid into rat bone marrow stromal cells (rBMSCs) to obtain a stable cell line with overexpression of PLAP-1 to verify whether PLAP-1 also acts as an inhibitory factor in rBMSCs during bone mineralization. A rBMSC line stably overexpressing PLAP-1 was established successfully as determined by the mRNA levels of PLAP-1, which were measured by real time-qPCR (RT-qPCR), and protein expression, which was measured by immunocytochemistry and western blot analysis. At the same time, a Cell Counting Kit-8 assay did not reveal any statistically significant changes in the transfected cells (P > 0.05). Then, mineral-inducing cultures were performed, and mineralized nodules were observed at weeks 2, 3 and 4 under a microscope. Alizarin Red (Sigma) staining was performed at 4 week to illustrate calcium accumulation. The mineralized nodules in the PLAP-1-transfected rBMSC group were fewer than those in the control groups. The time span of the formation of the mineralized nodules was prolonged. Meanwhile, osteogenic genes were also detected in the mineral-inducing cells by RT-qPCR. An RT-qPCR analysis demonstrated that the levels of the osteoblast markers of rBMSCs that were transfected with pBABE-hygro-PLAP-1, including Runx2, Osterix, alkaline phosphatase, bone sialoprotein and osteocalcin, were lower than those in the non-transfected rBMSCs and rBMSCs that were transfected with empty vector (P < 0.01). These results suggest that PLAP-1 has an inhibitory function in rBMSCs when they differentiate into osteoblast-like cells.

Effect of HIV-1 Tat on Secretion of TNF-α and IL-1β by U87 Cells in AIDS Patients with or without AIDS Dementia Complex

ObjectiveTo explore the role of HIV-1 tat gene variations in AIDS dementia complex (ADC) pathogenesis. MethodsHIV-1 tat genes derived from peripheral spleen and central basal ganglia of an AIDS patient with ADC and an AIDS patient without ADC were cloned for sequence analysis. HIV-1 tat gene sequence alignment was performed by using CLUSTAL W and the phylogentic analysis was conducted by using Neighbor-joining with MEGA4 software. All tat genes were used to construct recombinant retroviral expressing vector MSCV-IRES-GFP/tat. The MSCV-IRES-GFP/tat was cotransfected into 293T cells with pCMV-VSV-G and pUMVC vectors to assemble the recombinant retrovirus. After infection of gliomas U87 cells with equal amount of the recombinant retrovirus, TNF-α, and IL-1β concentrations in the supernatant of U87 cells were determined with ELISA. ResultsHIV-1 tat genes derived from peripheral spleen and central basal ganglia of the AIDS patient with ADC and the other one without ADC exhibited genetic variations. Tat variations and amino acid mutation sites existed mainly at Tat protein core functional area (38-47aa). All Tat proteins could induce U87 cells to produce TNF-α and IL-1β, but the level of IL-1β production was different among Tat proteins derived from the ADC patient's spleen, basal ganglia, and the non-ADC patient's spleen. The level of Tat proteins derived from the ADC patient's spleen, basal ganglia, and the non-ADC patient's spleen were obviously higher than that from the non-ADC patient's basal ganglia. ConclusionTat protein core functional area (38-47aa) may serve as the key area of enhancing the secretion of IL-1β. This may be related with the neurotoxicity of HIV-1 Tat.

Construction of retrovirol vector containing novel gene mgt -16 and its expression in mouse mesenchymal stem cells

Objective To construct the retroviral vector carrying novel gene mgt-16and to investigate its expression in mouse mesenchymal stem cells C3H/10T1/2(10T1/2).Methods DNA sequences containing mouse novel gene mgt-16was used as a template for PCR amplification of full length mgt-16cDNA.Then the DNA fragment was cloned into pEGFP-N1vector to produce pEGFP-N1-16vector after T-A cloning with pMD18Tplasmid and sequencing.The pEGFP-N1-16vector was confirmed by PCR,restriction enzyme digestion and sequencing analysis.The retroviral vector,pLEGFP-N1-16,was constructed using retroviral vector,pLEGFP-N1,and pEGFP-N1-16vector including mgt-16gene.The pLEGFPN1-16vector was verified by restriction enzyme digestion,sequenced,and then transfected into packaging cell line Phoenix to prepare EGFP fused mgt-16retrovirus particles,which were collected and used to infect 10T1/2cells.G418(400μg/mL)continuous selection was conducted to obtain 10T1/2cell clones stably overexpressing EGFP fused mgt-16.Fluorescence microscope was employed to determine the expression and subcellular localization of MGT-16in Phoenix and 10T1/2cells.Results A band of about 300bp size was observed by agarose gel electrophoresis after PCR amplification for mgt-16gene,and the result of sequencing showed that the sequence of insert fragment in T-A clones was identical to mgt-16gene reported in Genbank.PCR,restriction enzyme digestion and sequencing revealed that the pEGFP-N1-16plasmid was successfully constructed.Restriction enzyme digestion and sequencing revealed that the pLEGFP-N1-16plasmid was also successfully constructed.Phoenix and 10T1/2cells overexpressing MGT-16showed green fluorescence distribution in the cytoplasmic,especially around the perinuclear area.Conclusion We have successfully constructed a recombinant retroviral vector carrying novel gene,mgt-16,and expressed it in mouse mesenchymal stem cells,which provides a basis for studying the role of novel gene mgt-16in mesenchymal stem cells.

Effects of recombinant retroviral vector pLNCX2-human calcitonin gene-related peptide α on proliferation and osteogenic activity of human bone marrow mesenchymal stem cells

Objective To infect human bone marrow mesenchymal stem cells (hBMSCs) with recombinant retrovirus vector carrying human calcitonin gene-related peptide α (hCGRPα) gene in vitro,and observe its effects on the proliferation and osteogenic activity of hBMSCs.Methods The autologous primary hBMSCs were obtaind with Ficoll lymphocyte separation medium by gradient centrifugation,passaged and preserved.A recombinant retrovirus vector pLNCX2 carrying hCGRPα gene was constructed and infected into second generation of hBMSCs.The hBMSCs were divided into three groups:(1) experimental group:the pLNCX2-hCGRPα-infected hBMSCs; (2) empty vector group:empty vector pLNCX2-infected hBMSCs; (3) normal control group:cultured hBMSCs not given special treatment.At day 7,the mRNA and protein expression of hCGRPα was examined by using reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting respectively.The cell proliferation was determined by using methyl thiazoleterazolium (MTT) assay during 1 to 7 days.At day 14,the osteogenic activity of BMSCs was detected by using alkaline phosphatase (ALP) activity and osteocalcin in the cell culture medium.At day 7,the content of laminin (LN) was detected.Results Both the mRNA and protein expression of hCGRPα in hBMSCs was detectable in experimental group,but undetectable in empty vector group and normal control group.There was a higher cell proliferation rate in experimental group than in empty vector group (P ＜0.05) and normal control group (P ＜ 0.05).The ALP activity values,content of LN and osteocalcin in the cell culture medium were (119.33 ± 11.95) U/100 mg protein,(12.11 ± 1.56) μg/L and (1.123 ±0.115) μg/L in experimental group,which were significantly higher than those in empty vector group [(66.16 ±6.82) U/100 mg protein,(7.32 ±0.98) μg/L,(0.602 ±0.097) μg/L] and normal control group [(67.73 ± 7.31) U/100 mg protein,(6.46 ± 1.41) μg/L,(0.498 ± 0.109) μg/L] (P ＜ 0.05 for all),but there was no significant difference between empty vector group and normal control group (P ＞ 0.05).Conclusion The recombinant retrovirus hCGRPα was successfully transferred into hBMSCs.The hCGRPα gene could be expressed in hBMSCs,promote the cell proliferation and its osteogenic activity. Key words: Recombinant retrovirus; Calcitonin gene-related peptide; Bone marrow mesenchymal stem cells; Cell proliferation; Osteogenic activity

Broadening the Indications for Hematopoietic Stem Cell Genetic Therapies

The use of recombinant retroviral vectors to effect corrective genetic therapies in hematopoietic stem cells (HSCs) has long been predicted to revolutionize medicine. Two recent papers in Science now show that this technology could be considered as effective as, and perhaps superior to, allogeneic HSC transplants in some rare diseases.