Lentiviral Vector-mediated RNA Interference Research Articles

Control of mouse graft-versus-host disease following allogeneic bone marrow transplantation by blocking the CD28/B7 signaling pathway with lentiviral vector-mediated RNA interference

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is an effective way to cure hematological malignancies. However, graft-versus-host disease (GVHD) following transplantation limits the clinical application to some extent. The donor T lymphocytes play a central role in the occurrence and development of GVHD. Control of GVHD by inhibition of T cell proliferation by blocking the CD28/B7 signaling pathway with RNA interference has not been examined. In this study, we constructed a lentiviral vector carrying CD28 shRNA and generated genetically engineered splenocytes through transduction in a murine allogeneic bone marrow transplantation model. The survival and the occurrence of GVHD in transplanted mice were monitored every day. Liver, intestine, skin, and other tissues from the mice in each group were used for histological examination. We also determined plasma concentrations of interleukin (IL)-2, IL-4, IL-5, IL-10, IL-13, and interferon gamma (IFN-γ). Recipient bone marrow from mice that had survived for an extended period was examined to detect chimerism. We succeeded in suppressing the expression of CD28 gene and controlling mouse GVHD following allogeneic bone marrow transplantation in the engineered spleen cell group. These suggest that blocking the CD28/B7 signal transduction pathway with lentiviral vector-mediated RNA interference effectively controlled the occurrence of mouse GVHD following allogeneic bone marrow transplantation. Its mechanism could be due to the inhibition of T cell proliferation and, simultaneously, the promotion of the differentiation of TH0 to TH2 cells, thereby reducing GVHD in the mouse transplantation model.

APC/C-Cdh1 as a Novel Regulator of Hematopoietic Stem Cell Differentiation.

AbstractAbstract 1563 Introduction:The anaphase-promoting complex/cyclosome (APC/C) is an E3 ubiquitin ligase that regulates cell cycle progression. This is achieved by targeting various cell cycle regulators for proteasomal destruction. APC/C in conjunction with its adaptor protein Cdh1, both stabilizes G1-Phase (a pre-condition for an accurate cell cycle progression) and is involved in the induction of cell cycle arrest and differentiation. Further evidence suggests that Cdh1 is involved in the differentiation of a variety of cells such as neurons, myocytes, hepatocytes and lens epithelial cells. During differentiation Cdh1 interacts with the TGFb signaling pathway, targets Id2 for destruction and indirectly leads to accumulation of p27 by Skp2 degradation. We have been able to demonstrate that the expression of Cdh1 is decreased in both, AML cell lines and in primary blast samples carrying the translocation t(8;21), which consequently leads to an AML1/Eto fusion protein and is one of the most common chromosomal rearrangements in AML. Furthermore, we have data suggesting that APC/C-Cdh1 significantly influences the differentiation of malignant myeloid cells. Here, we have analyzed the expression of Cdh1, its target proteins and relevant cell cycle regulators during normal myeloid differentiation. Methods:A cytokine cocktail consisting of SCF (50ng/ml), IL-3 (5 ng/ml) and G-CSF (100 ng/ml) was used to induce differentiation of CD34+ cells into CD11b+ macrophage-like cells over seven days. Daily protein isolation, CD11b-FACS and FACS analysis of propidium iodide staining were performed to analyze Cdh1 status, differentiation kinetics and cell cycle distribution. In addition, we have established a Cdh1 knockdown in CD34+ cells by lentiviral vector mediated RNA interference. By means of GFP-cell-sorting the initially achieved transduction efficiency of 30% in CD34+ cells was increased to 70–80%. Results:The differentiation experiments carried out with normal CD34+ cells showed that after 7 days of stimulation the predominant majority of them had lost the CD34 marker and about 30% expressed CD11b on their surface confirming previous results. During the differentiation process an initial rise in Cdh1 levels, followed by a continuously high expression, was observed. Furthermore, we detected a downregulation of the Cdh1 target proteins Id2 and Skp2 and stable protein levels of p27. The cell cycle profile indicated an initial proliferation with an incremental G2/M-peak and at day 6 increasing apoptosis with a high Sub-G1-peak. In the transduced CD34+ cells we have been able to confirm by Western blotting analysis and RT-PCR that the CD34 positive cells harboring a Cdh1 shRNA had significantly decreased protein and RNA levels of Cdh1 compared to CD34 positive cells harboring a control shRNA against GFP. Analysis of the influence of Cdh1 knockdown on differentiation of CD34+ cells is ongoing and will be presented at the meeting. Conclusion:These results are consistent with the important role of Cdh1 in initiating differentiation and also show its sustained function in post mitotic myeloid cells. Studying the differentiation characteristics of CD34+ cells with a Cdh1 knockdown is likely to help to further determine its function. Disclosures:No relevant conflicts of interest to declare.

Downregulation of the Cell-Cycle Regulating Ubiquitin-Ligase APC/CCdh1 May Contribute to the Differentiation Block of AML1/Eto Positive AML.

Abstract 5045 IntroductionThe anaphase-promoting complex/cyclosome (APC/C) is an E3 ubiquitin ligase regulating cell cycle progression by targeting various cell cycle regulators for proteasomal degradation. It is activated by the adaptor proteins Cdc20 in mitosis and by Cdh1 in late mitosis and G1/G0. Thereby, Cdh1 establishes a stable G1 phase enabling the cell to either exit the cell cycle and differentiate or to prepare for a new round of cell division. It has also been shown that Cdh1 plays a role in the differentiation of various cell types, such as neurons, myocytes, hepatocytes and lens epithelial cells. Methods and ResultsWe have examined the regulation of Cdh1 in several acute myeloid leukemia (AML) cell lines. We found that in the AML1/Eto positive leukemia cell lines SKNO-1 and Kasumi-1, Cdh1 protein and RNA levels are lower than in AML1/Eto negative cell lines KG-1 and HL-60. In addition, Cdh1 protein level in an AML1/Eto positive primary blast sample was lower than in AML1/Eto negative patient samples. The translocation t(8;21) is one of the most frequent chromosomal rearrangement in AML and results in an AML1/Eto fusion protein, which can act as a transcriptional repressor. Thus, our results are consistent with AML1/Eto mediated downregulation of Cdh1. To evaluate the potential role of APC/CCdh1 in myeloid differentiation, we established a stable Cdh1 knockdown (kd) in the AML1/Eto negative HL60 cell line with high Cdh1 expression by lentiviral vector mediated RNA interference. HL60 cells harbouring either a Cdh1 shRNA or a control shRNA against GFP were established simultaneously. We used PMA at concentrations of 0.5, 1, 2 and 50 nM to differentiate these cells into CD11b positive macrophage-like cells over 48h. Protein isolation and analysis of CD11b expression by flow cytometry were performed at 0, 6h, 12h, 24h and 48h to examine differentiation kinetics. Cdh1 and target proteins with a potential role in cell cycle arrest and differentiation, such as Skp2 (an activator of the SCF-ubiquitin ligase targeting p21 and p27) and ID2 (inhibitor of differentiation 2), were analyzed by Western blotting. We observed that kd of Cdh1 in HL60 cells resulted in 10% to 20% lower CD11b expression at any time, when PMA was used at concentrations 0, 0.5, 1nM over 48h. ID2 and Skp2 were stabilized in these Cdh1 kd cells compared to the control correlating with the less differentiated state. In addition, HL60 cells with a stable Skp2 kd showed a higher CD11b expression indicating a more differentiated status compared to the control. ConclusionThis is the first report that indicates a role for APC/CCdh1 in the differentiation of myeloid cells with SCFSkp2 being one of the relevant targets. Downregulation of Cdh1 may contribute to the differentiation block of AML1/Eto postive AML. DisclosuresNo relevant conflicts of interest to declare.

Lentiviral vector-mediated RNA interference of HBs gene inhibits replication of HBV

Objective:To construct a lentiviral vector(LV) of RNA interference(RNAi) targeting HBs gene and to observe its effect on the replication of HBV and expression of antigens.Methods: The effective sequence of siRNA targeting HBs gene was confirmed in our previous study.The complementary DNA containing both sense and antisense Oligo DNA of the targeting sequence was designed,synthesized and cloned into the lentivirus vector(pGCLM-GFP).The resulting lentivirus vector containing HBs shRNA was named LVshHBs,and it was confirmed by PCR and sequencing.293T cells were cotransfected with lentivirus vector pGCLM-GFP,pHelper 1.0 and pHelper 2.0.All virus stocks were produced by calcium phosphate-mediated transfection.The titer of virus was tested according to the expression level of GFP.HepG2.2.15 cells were infected with LVshHBs and the supernatant of the cells was subjected to ELISA,Western blotting analysis and HBV DNA quantitative analysis.Results: PCR and DNA sequencing demonstrated that the LVshHBs-producing HBs shRNA was successfully constructed.The titer of concentrated virus was 5×108-2×109 TU /ml.The inhibitory effect was efficient and the corresponding viral transcript and expression of antigens were decreased after infection.The inhibitory effect was observed 4 days after infection and peaked 9 days after the initial treatment with RNAi.Secreted HBsAg was reduced by 70% in cell culture compared with the negative control,which is also confirmed by Western blotting and real-time PCR.After quantification of HBV DNA,the level of DNA relative to the controls was also significantly reduced after RNAi treatment(P0.05).Conclusion: The lentivirus RNAi vector of HBs has been successfully constructed.The lentiviral microRNA-based RNAi targeting HBs can specifically mediate the down-regulation of HBs expression,inhibiting HBV replication and antigen expression.

Combined antiviral activity of interferon-α and RNA interference directed against hepatitis C without affecting vector delivery and gene silencing

The current standard interferon-alpha (IFN-α)-based therapy for chronic hepatitis C virus (HCV) infection is only effective in approximately half of the patients, prompting the need for alternative treatments. RNA interference (RNAi) represents novel approach to combat HCV by sequence-specific targeting of viral or host factors involved in infection. Monotherapy of RNAi, however, may lead to therapeutic resistance by mutational escape of the virus. Here, we proposed that combining lentiviral vector-mediated RNAi and IFN-α could be more effective and avoid therapeutic resistance. In this study, we found that IFN-α treatment did not interfere with RNAi-mediated gene silencing. RNAi and IFN-α act independently on HCV replication showing combined antiviral activity when used simultaneously or sequentially. Transduction of mouse hepatocytes in vivo and in vitro was not effected by IFN-α treatment. In conclusion, RNAi and IFN-α can be effectively combined without cross-interference and may represent a promising combinational strategy for the treatment of hepatitis C.

Functional analysis of the quantitative expression of a costimulatory molecule on dendritic cells using lentiviral vector-mediated RNA interference

The increasing number of co-stimulatory molecules identified on dendritic cells (DC) to date highlights the complex regulation of co-stimulatory signals in T cell activation. We previously established a single lentiviral vector system to stably express short hairpin RNA (shRNA) to induce RNA interference (RNAi) in cell lines and primary T cells. We reasoned that the choice of shRNA target sequences in the lentiviral vector system would also allow us to regulate different levels of surface expression of a co-stimulatory molecule stably and reproducibly. In this study, we first demonstrated that lentiviral vectors delivered RNA interference in DC without functional impairments. We used CD40 as a target co-stimulatory molecule to demonstrate the feasibility of using lentiviral vectors in delivering different shRNA target sequences to genetically modify DC that expressed different levels of CD40. We provided functional data to further demonstrate that quantitative expression of CD40 on LPS-stimulated DC have different functional outcomes on Ag-specific T cell responses in vitro. Collectively, we developed a simple system that will allow us to examine functional significance(s) of the quantitative and/or qualitative expression of a single or multiple co-stimulatory molecule(s) on DC.

Strong inducible knockdown of APC/CCdc20 does not cause mitotic arrest in human somatic cells

The anaphase-promoting complex/cyclosome (APC/C) is a conserved ubiquitin ligase controlling mitosis and G1 phase of the cell cycle. The APC/C is activated by two regulatory subunits Cdc20 (APC/CCdc20) and Cdh1 (APC/CCdh1) to target securin, mitotic cyclins and other cell cycle regulatory proteins. Cdc20 is essential for sister chromatid separation at the meta- to anaphase transition in yeast, drosophila and perhaps mouse embryos. However, whether Cdc20 is essential for mitotic control of human somatic cells is uncertain. Therefore, we used a lentiviral vector-mediated inducible RNA interference (RNAi) system to generate strong downregulation of Cdc20 expression in clonal cells to further elucidate the role of human Cdc20. Here we show, that even an almost complete knockdown of Cdc20 below the detection limit in western blots does neither cause a mitotic block nor significant stabilization of the APCCdc20 substrates cyclin B and securin. Thus, there may be redundant mechanisms of mitotic control in the human somatic cell cycle.

Lentivirus-Mediated RNA Interference Targeting Vascular Endothelial Growth Factor Gene Enhances the Sensitivity of K562 Cells to Imatinib

Background: Vascular endothelial growth factor (VEGF), one of the most potent mediators of angiogenesis, plays an important role in tumor cell proliferation and migration. Elevated VEGF levels are found in both the plasma and the bone marrow of patients with chronic myelogeneous leukemia(CML). Moreover, the CML-associated oncogene Bcr-Abl induces endogenous secretion of VEGF. The aim of this study was to construct a lentiviral vector-mediated RNA interference (RNAi) of vascular endothelial growth factor (VEGF) gene and to investigate the effects of RNAi inhibiting VEGF gene alone or in combination with imatinib on the proliferation and apoptosis of K562 leukemic cell line.Methods: A lentiviral expression vector containing short hairpin RNA (shRNA ) targeting VEGF was constructed and cotransfected with the pPACKH1 packaging plasmids mixture into 293T cells by Lipofectamine 2000. K562 cells were infected with the packaged lentivirus. The levels of VEGF mRNA and protein were detected by real-time quantitative RT- PCR, Western blot and ELISA. Cellular proliferation was determined by trypan blue exclusion and MTT assay. imatinib -induced apoptosis was analyzed by flow cytometry.Results: The lentiviral shRNA vector targeting VEGF has been constructed and transfected into K562 cells successfully. The expression of VEGF mRNA and protein in K562-shVEGF cells transfected with pRNAT-shRNA were significantly inhibited when compared with those of K562 and K562–con cells (mock transfection). The proliferation rate of K562-shVEGF cells slowed down. After imatinib treatment, the percentages of apoptotic cells in K562-shVEGF cells increased more significantly than those of K562 and K562–con cells.Conclusion: Suppression of VEGF by lentivirus-mediated RNAi could effectively inhibit proliferation and increase the sensitivity of K562 cells to imatinib. These results indicate that Bcr/Abl-targeting compound in combination with anti-angiogenic therapies may provide a potent therapeutic strategy to CML.

Inhibitory effects of lentiviral vector-mediated RNA interference on proliferation-inducing ligand expression in human pancreatic cancer <I>in vitro</I>

Inhibitory effects of lentiviral vector-mediated RNA interference on proliferation-inducing ligand expression in human pancreatic cancer <I>in vitro</I>

Inhibition of the Replication of Feline Iommunodeficiency Virus by Lentiviral Vector-Mediated RNA Interference in Feline Cell Lines

RNA interference (RNAi) is a sequence-specific RNA degradation process. To inhibit feline immunodeficiency virus (FIV) replication by RNAi, we generated a lentiviral vector expressing a short hairpin RNA (shRNA) that targeted the gag gene of FIV (shGag). shGag transfer significantly inhibited viral replication in cell lines that were chronically infected with FIV, i.e., the 3201/UK8 low, 3201/UK8 high, FL4, and CRFK/FIV cell lines. Moreover, 3201 cells were transduced with the lentiviral vectors and then inoculated with FIV. Although the amount of FIV proviral DNA in shGag-transduced cells was similar to that in the cells transduced with unrelated shRNA or mock-transduced cells, the amount of reverse transcriptase (RT) activity was significantly reduced in the culture supernatant of shGag-expressing cells from 15 to 27 days after inoculation. Thirty days after inoculation, no significant difference was observed in the RT activities but virus with a mutation in the target region of shGag was detected in approximately 21% of the replicated viruses. Therefore, abolishment of the silencing effect of shGag may be due to reasons other than the emergence of escape mutants. These results are useful for developing an RNAi-based gene therapy strategy for controlling FIV infection.

A Partial Down-regulation of WASP Is Sufficient to Inhibit Podosome Formation in Dendritic Cells

The Wiskott Aldrich syndrome protein (WASP) is a hematopoietic-specific cytoskeletal regulator that is necessary for induction of normal immunity. In the context of effective gene therapy for WAS, cellular models of human WASP deficiency are important for definition of the threshold of protein expression required for optimal activity. Using lentiviral vector-mediated RNA interference (RNAi), we were able to down-regulate the levels of human WASP in cell lines and primary cells. In dendritic cells (DC), RNAi-induced WASP deficiency did not impair phenotypic maturation but perturbed cytoskeletal organization. As a result, podosomes, which are actin-rich structures present in immature adherent DC, were formed less efficiently and motility was disturbed. Overall, treatment of cells with RNAi recapitulated the phenotype of cells derived from patients or animals with inactivating mutations of the WAS gene. Interestingly, reduction of the levels of WASP to about 60% of normal was sufficient to inhibit the formation of podosomes in DC, implying that this cell type requires near-normal levels of WASP to sustain physiological cytoskeleton-dependent activities.

Hypoxia-induced resistance to cisplatin and doxorubicin in non-small cell lung cancer is inhibited by silencing of HIF-1α gene

Hypoxia is associated with human non-small cell lung cancers (NSCLC), which are highly resistant to chemotherapy. The hypoxia inducible factor (HIF) as a transcription factor in response to hypoxia indicates that it could be a novel, tumor-specific target for anticancer therapy. We hypothesized that disruption of HIF pathway through lentiviral vector-mediated HIF-1alpha RNA interference (RNAi) could reverse the hypoxia-induced resistance to chemotherapy. We transfected Human NSCLC cell lines, SPCA1 and A549 with HIF-1alpha specific RNAi lentiviral vectors as well as controls. HIF-1alpha silenced cells [SPCA1/HIF-1alpha(-) and A549/HIF-1alpha(-)] were screened by blasticidin. They were incubated in 19 or 0.5% O2 for 16 h followed by the assessment of chemosensitivity to cisplatin and doxorubicin with MTT and clonogenic assays. Quantitative RT-PCR and Western blot analysis were used to detect the expressions of HIF-1alpha mRNA and protein, respectively. Moreover, flow cytometry was used to monitor the expression of P-glycoprotein. Exposure of SPCA1 and A549 cells to 0.5% O2 significantly increased resistance to cisplatin and doxorubicin, in contrast to cells incubated in normoxia. Transduction of SPCA1 with HIF-1alpha RNAi vector resulted in sequence specific silencing with 87.2 and 84.6% decreases of HIF-1alpha mRNA transcription and 97.3 and 94.8% of protein expressions in normoxia and hypoxia, respectively. Correspondingly, they are 89.2, 89.9% and 97.2, 88.4% decreases in A549 cells. Hypoxia-induced resistance to cisplatin and doxorubicin were reversed in SPCA1/HIF-1alpha(-) and A549/HIF-1alpha(-) cells. There was no significant P-glycoprotein increase induced by hypoxia in NSCLC cells. Our studies demonstrated that hypoxia-induced chemoresistance to cisplatin and doxorubicin in NSCLC cells is through the HIF pathway. MDR1 regulation may not be involved in hypoxia-induced chemoresistance. Combining delivery of HIF-1alpha RNAi lentiviral vector with cisplatin-related chemotherapy regimens may enable us to develop more effective strategy for NSCLC therapy.

P53 Silencing by Lentiviral Vector Mediated RNA Interference in Murine Hematopoietic Cells and the Development of a Myelogenous Proliferative Disease.

The ability for siRNA containing lentiviral vectors to specifically downregulate gene expression in the hematopoietic system following hematopoietic stem cell transplantation provides a new tool for developing disease models of leukemia and other hematological disorders. The tumor suppressor gene, p53, has been implicated in the genesis or progression of hematological malignancies, such as AML, MDS, and CML, and disorders such as myeloproliferative disease (MPD). Following transduction of Ly5.1 hematopoietic stem cells using a lentiviral vector expressing small interfering RNAs specific for mouse p53, cells were transplanted into lethally irradiated syngeneic recipient mice. Peripheral blood analyses by FACS at 7 weeks posttransplant showed significantly increased numbers of Gr1 Mac1 double positive progenitor like cells in 4 of 4 mice (73±14% compared to control siGFP transplanted mice 5±0.5%). Interestingly, 3 of 4 transplanted mice showed signs of morbidity by 16 weeks posttransplant due to a myelogenous proliferative disease (MPD) phenotype characterized by high peripheral blood neutrophil counts (>70% of white blood cells) and anemia (<106 RBC/μL), and extensive neutrophil infiltration into spleen and liver. Furthermore, peripheral blood smears show an expansion of megakaryocytes. Bone marrow mononuclear cells from transplanted mice were analyzed for p53 protein expression following p53 stabilization by irradiation and significantly lower levels of p53 were detected in sip53-transduced mice compared to siGFP lentiviral vector control mice. No altering of normal hematopoietic cell differentiation was observed in control mice from nonspecific RNAi interactions, as determined by FACS or blood cell count analyses. These results demonstrate that downregulated expression of p53 in mice hematopoietic cells yields a myelogenous proliferative disease and creates a model for the study of p53 involvement in the genesis and progression of hematological disorders. Furthermore, these results demonstrate the ability of lentiviral vector mediated siRNA to efficiently downregulate endogenous gene expression in vivo and model human disease.