Lentiviral Gene Delivery System Research Articles

Paired-related homeobox 1 induces epithelial-mesenchymal transition in oesophageal squamous cancer.

It is unclear that paired-related homeobox 1 (PRRX1) induces epithelial-mesenchymal transition (EMT) in oesophageal cancer and the specific function of PRRX1 in oesophageal cancer metastasis. To assess the significance of PRRX1 expression and investigate the mechanism of EMT in oesophageal cancer metastasis. Detect the expression of PRRX1 by immunohistochemistry in oesophageal tumour tissues and adjacent normal oesophageal tissues; the PRRX1 short hairpin RNA (shRNA) or blank vector lentiviral gene delivery system was transfected into cells; cell proliferation assay, soft agar colony formation assays, cell invasion and migration assays and animal studies were used to observe cells biological characteristics In vitro and in vivo; XAV939 and LiCl were used to alter the activity of Wnt/β-catenin pathway. Immunofluorescence staining and western blot analysis were used to detect protein expression of EMT markers and Wnt/β-catenin pathway. PRRX1 is expressed at high levels in oesophageal cancer specimens and is closely related to tumour metastasis in patients with oesophageal cancer. Regulation of PRRX1 expression might exert obvious effects on cell proliferation, especially the migration and invasion of oesophageal cancer cells. Moreover, silencing PRRX1 expression using a shRNA produced the opposite effects. In addition, when PRRX1 was overexpressed, inhibition of the Wnt/β-catenin pathway with XAV939 negated the effect of PRRX1 on EMT, whereas when PRRX1 was downregulated, activation of the Wnt/β-catenin pathway with LiCl impaired the effect on EMT. PRRX1 is upregulated in oesophageal cancer is closely correlated with cancer metastasis. Additionally, PRRX1 induces EMT in oesophageal cancer metastasis through activation of Wnt/β-catenin signalling.

Expression of WNT genes in cervical cancer-derived cells: Implication of WNT7A in cell proliferation and migration

According to the multifactorial model of cervical cancer (CC) causation, it is now recognized that other modifications, in addition to Human papillomavirus (HPV) infection, are necessary for the development of this neoplasia. Among these, it has been proposed that a dysregulation of the WNT pathway might favor malignant progression of HPV-immortalized keratinocytes. The aim of this study was to identify components of the WNT pathway differentially expressed in CC vs. non-tumorigenic, but immortalized human keratinocytes. Interestingly, WNT7A expression was found strongly downregulated in cell lines and biopsies derived from CC. Restoration of WNT7A in CC-derived cell lines using a lentiviral gene delivery system or after adding a recombinant human protein decreases cell proliferation. Likewise, WNT7A silencing in non-tumorigenic cells markedly accelerates proliferation. Decreased WNT7A expression was due to hypermethylation at particular CpG sites. To our knowledge, this is the first study reporting reduced WNT7A levels in CC-derived cells and that ectopic WNT7A restoration negatively affects cell proliferation and migration.

Abstract 2973: Mouse models of lung cancer mediated by lentiviral gene delivery

Abstract Lung cancer, the leading cause of cancer deaths, is responsible for 1.4 million lives lost worldwide every year. Good animal models that can faithfully recapitulate the human disease are in great need for pre-clinical studies. Based on the different responses to treatment, lung cancer is divided into two major classes: small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). Adenocarcinoma (AdC) and squamous cell lung cancer (SqCC) are two major sub-types of NSCLC. We have successfully developed a novel lentiviral gene delivery system to study the initiation and development of all these lung cancers. We used CA2Cre-shp53 lentiviral vector to initiate lung adenocarcinoma in LSL-KrasG12D mice and proved the importance of NF-κB/IKK2 pathway in tumor cell proliferation. To search for novel tumor suppressors in lung cancer, we incorporated an shRNA library targeting ∼5000 cell signal genes into our CA2Cre lentivector and did the screening in the mice. We have identified several dozens of candidates and are validating these genes back in animals. SCLC is the most malignant form of lung cancer with a five-year survival less than 6%. A mouse model with p53/Rb deletions activated by Adeno-Cre has been established in Berns lab. To improve the model and study other genes that are frequently mutated in human SCLC, we designed a single lentiviral vector to deliver oncogenes (L-myc etc.) and shRNAs against tumor suppressors (p53, Rb etc) and successfully initiated SCLC in wild-type mice. Furthermore, we included luciferase or fluorescence protein genes in the same vector to follow tumor growth and metastasis in the live animal. Using this new model, we are now studying the crosstalk of multiple mutations in the SCLC development. SqCC has not been well modeled by genetic approaches. Interestingly, when we gave KrasG12D-shp53 lentiviral vector after lung tissue damage induced by naphthalene, the mice quickly developed squamous tumors in the big bronchi other than adenocarcinomas in the peripheral lung. Kras mutations are actually not common in human SqCC patients, so we are now optimizing the model and see if more relevant genetic changes such FGFR1 amplification can also induce this type of lung cancer in mice. We hope that our unique lentiviral tool can simplify and accelerate the lung cancer study to understand the big signal pathway complex leading to tumorigenesis and metastasis in the lung and search for new therapies for this deadly human disease. Citation Format: Yifeng Xia, Narayana Yeddula, Eugene Ke, Mathias Leblanc, Inder Verma. Mouse models of lung cancer mediated by lentiviral gene delivery. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2973. doi:10.1158/1538-7445.AM2014-2973

Abstract PR3: NSCLC and SCLC mouse models mediated by lentiviral gene delivery

Abstract Lung cancer, the leading cause of cancer deaths, is responsible for 1.3 million lives lost worldwide every year. Good animal models that can faithfully recapitulate the human disease are in great need for pre-clinical studies. Based on the different responses to treatment, lung cancer is divided into two major classes: small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). We have developed a novel lentiviral gene delivery system to study the initiation and development of both types of lung cancer. We use lentiviral vectors to establish lung tumors in mice for the following reasons: 1) lentiviruses infect almost any type of cells, and transgene expression can be controlled by a tissue-specific promoter, which allows a more precise tracing of the origin of the cancer cell; 2) lentiviruses integrate into genomic DNA so that it is possible to stably deliver oncogenes and shRNAs against tumor suppressors, and bypass the requirement of numerous conventional genetic crossings; 3) viral titers can be controlled so as to infect only a few cells, in order to more precisely recapitulate human cancer initiation. We used CA2Cre-shp53 lentiviral vector to initiate lung adenocarcinoma in KrasG12D mice and studied the role of NF-κB/IKK2 in tumor development. We found that IKK2 depletion in tumor cells significantly attenuated tumor proliferation and significantly prolonged mouse survival. We identified Timp-1 (a tissue inhibitor of metalloproteinases), one of the NF-κB target genes, as a key mediator for tumor growth. Timp-1 binds to its receptor CD63, activates Erk signaling pathway and stimulates cell proliferation. Knockdown of either IKK2 or Timp-1 by shRNAs reduced tumor growth in both xenograft and lentiviral models, suggesting the possible application of IKK2 and Timp-1 inhibitors in treating lung cancer. Due to the safety concerns of systemic NF-κB inhibition, IKK2 inhibitors haven't been applied to the clinical trials. Our identification of Timp-1 may serve as an alternative target for blocking tumor proliferation. Timp-1 neutralizing antibodies are currently being tested to treat mice carrying Kras adenocarcinomas. SCLC is the most malignant form of lung cancer with a five-year survival less than 6%. A mouse model with p53/Rb deletions activated by Adeno-Cre has been established in Berns lab. To improve the model and study other genes that are frequently mutated in human SCLC, we designed a single lentiviral vector to deliver oncogenes (L-myc) and shRNAs against tumor suppressors (p53, Rb etc) and successfully initiated SCLC in wild-type mice. Furthermore, we included luciferase or fluorescence protein genes in the same vector to follow tumor growth and metastasis in the live animal. Using this new model, we are now studying the crosstalk of multiple mutations in the SCLC development. We hope that our unique lentiviral tool can simplify and accelerate the lung cancer study to understand the big signal pathway complex leading to tumorigenesis in the lung and search for new therapies for this deadly human disease. This abstract is also presented as Poster A45.

Development of BMP7-producing human cells, using a third generation lentiviral gene delivery system

Bone morphogenetic protein 7 (BMP7), a member of the transforming growth factor β (TGF-β) superfamily, plays important roles in the development of various tissues and organs in mouse and human. In particular, BMP7 is critical for the formation of the nervous system and it is considered to have therapeutic potential in brain injury and stroke. One approach to make BMP7 more suitable for therapeutic purposes is the development of efficient vectors that allow the consistent, reliable and cost-effective production of the BMP7 protein.In this study, we developed an efficient BMP7 delivery system, using a third generation lentiviral vector to produce functional BMP7 protein. The lentiviral transduction of several human cell types, including human embryonic kidney 293 (HEK293) cells, amniotic fluid cells, NTera2 neurons (NT2-N) and primary neuronal cultures resulted in BMP7 expression. The production of BMP7 protein was achieved for at least 4 weeks post-transduction, as determined by enzyme-linked immunosorbent assay (ELISA). SMAD phosphorylation and neuronal differentiation assays verified the bioactivity and functionality of the lentiviral-based BMP7 protein, respectively. In addition, the intracerebroventricular injection of the lentivirus resulted in exogenous BMP7 expression in both neurons and astrocytes in the mouse brain.Taken together, this gene delivery system provides a reliable source of functional BMP7 protein for future in vitro and in vivo studies.

Parkinson-related parkin reduces α-Synuclein phosphorylation in a gene transfer model

Backgroundα-Synuclein aggregates in Lewy bodies and plays a central role in the pathogenesis of a group of neurodegenerative disorders, known as "Synucleinopathies", including Parkinson's disease. Parkin mutations result in loss of parkin E3-ubiquitin ligase activity and cause autosomal recessive early onset parkinsonism.ResultsWe tested how these two genes interact by examining the effects of parkin on post-translational modification of α-Synuclein in gene transfer animal models, using a lentiviral gene delivery system into the striatum of 2-month old male Sprague Dawley rats.Viral expression of wild type α-Synuclein caused accumulation of α-Synuclein and was associated with increased cell death and inflammation. α-Synuclein increased PLK2 levels and GSK-3β activity and increased the levels of phosphorylated α-Synuclein and Tau. Parkin co-expression reduced the levels of phosphorylated α-Synuclein and attenuated cell death and inflammation. Parkin reduced PLK2 levels and increased PP2A activation.ConclusionsThese data suggest that parkin reduces α-Synuclein levels and alters the balance between phosphatase and kinase activities that affect the levels of phosphorylated α-Synuclein. These results indicate novel mechanisms for parkin protection against α-Synuclein-induced toxicity in PD.

Highly Efficient Stable Expression of Indoleamine 2,3 Dioxygenase Gene in Primary Fibroblasts

Indoleamine 2,3 dioxygenase (IDO) is a potent immunomodulatory enzyme that has recently attracted significant attention for its potential application as an inducer of immunotolerance in transplantation. We have previously demonstrated that a collagen matrix populated with IDO-expressing fibroblasts can be applied successfully in suppressing islet allogeneic immune response. Meanwhile, a critical aspect of such immunological intervention relies largely on effective long-term expression of the IDO gene. Moreover, gene manipulation of primary cells is known to be challenging due to unsatisfactory expression of the exogenous gene. In this study, a lentiviral gene delivery system has been employed to transduce primary fibroblasts. We used polybrene to efficiently deliver the IDO gene into primary fibroblasts and showed a significant increase (about tenfold) in the rate of gene transfection. In addition, by the use of fluorescence-activated cell sorting, a 95% pure population of IDO-expressing fibroblasts was successfully obtained. The efficiency of the IDO expression and the activity of the enzyme have been confirmed by Western blotting, fluorescence-activated cell sorting analysis, and Kynurenine assay, respectively. The findings of this study revealed simple and effective strategies through which an efficient and stable expression of IDO can be achieved for primary cells which, in turn, significantly improves its potential as a tool for achieving immunotolerance in different types of transplantation.

An Internal Polyadenylation Signal Substantially Increases Expression Levels of Lentivirus-Delivered Transgenes but Has the Potential to Reduce Viral Titer in a Promoter-Dependent Manner

In lentiviral gene delivery systems, transgene expression cassettes are commonly cloned without a polyadenylation signal to prevent disruption of full-length lentiviral genomes on mRNA maturation in producer cells. The lack of the polyadenylation signal, however, has the potential to reduce stability and translation efficiency of transgene mRNA. Therefore, we have assessed the effect of a strong internal polyadenylation [poly(A)] signal on both transgene expression levels in virus-infected cells and functional viral titer, in a series of eight self-inactivating lentiviruses expressing the mOrange transgene under the control of the constitutive cytomegalovirus (CMV), elongation factor 1alpha (EF1alpha), and beta-actin promoters or the highly tissue-specific prostate-specific antigen/probasin hybrid (PSA/Pb) promoter with or without a simian virus 40 (SV40) early polyadenylation signal downstream of the mOrange-coding sequence. We show that mOrange expression levels in virus-infected HEK-293, LNCaP, and primary prostate epithelial cells were increased 3- to 6.5-fold when an internal polyadenylation signal was present. When the CMV and EF1alpha promoters were used, functional viral titer decreased 8- to 9-fold in the presence of the polyadenylation signal, but titer was not affected when transgene expression was driven by the beta-actin promoter or tissue-specific PSA/Pb promoter. We therefore conclude that an internal polyadenylation signal in lentiviral vectors has a highly beneficial effect on transgene expression, but reduces viral titer in a promoter-dependent manner.

10. Developing a lentiviral gene delivery system in stem cells for the treatment of Sanfilippo syndrome (MPS IIIA)

10. Developing a lentiviral gene delivery system in stem cells for the treatment of Sanfilippo syndrome (MPS IIIA)

Efficiency of the Elongation Factor-1α Promoter in Mammalian Embryonic Stem Cells Using Lentiviral Gene Delivery Systems

The establishment of new technology for genetic modification in human embryonic stem (ES) cell lines has raised great hopes for achieving new ground in basic and clinical research. Recently, lentiviral vector technology has been shown to be highly effective and therefore could emerge as a popular tool for human ES cell genetic modification. The objectives of this study were to evaluate the efficiency of promoters in lentiviral gene delivery systems in mammalian ES cells, including mouse, monkey, and human, and to construct efficient and optimized conditions for lentivirus-mediated transfection systems. Mammalian ES cells were transfected with self-inactivating (SIN) human immunodeficiency virus type-1 (HIV-1)-based lentiviral vectors containing the human polypeptide chain elongation factor-1alpha (EF-1alpha) promoter or cytomegalovirus (CMV) promoter and analyzed by fluorescence-activated cell sorting (FACS) analysis for the expression of the enhanced green fluorescent protein (eGFP) reporter gene. The efficiency of the EF-1alpha promoter was higher than that of the CMV promoter in all ES cells tested. The EF-1alpha promoter efficiently drove gene expression (14.74%) compared with CMV promoter (3.69%) in human ES cells. We generated a stable eGFP+ human ES cell line (CHA3-EGFP human ES cells) that continuously expressed high levels of EGFP ( approximately 95%) from the EF-1alpha promoter and was maintained for up to 60 weeks with undifferentiated proliferation. The established CHA3-EGFP human ES cell lines were characterized as being negative for nondifferentiation markers and teratoma formation. These results imply that genetic modification by lentiviral vectors with specific promoters in ES cells constitute a powerful tool for guided differentiation as well as gene therapy.

Use of a lentiviral gene delivery system to target GnRH neurons

Use of a lentiviral gene delivery system to target GnRH neurons

Cell Type–Specific Targeting with Sindbis Pseudotyped Lentiviral Vectors Displaying Anti-CCR5 Single-Chain Antibodies

Lentiviral vectors are among the most efficient tools for gene delivery into mammalian cells. A major goal of lentiviral gene delivery systems is to develop vectors that can efficiently target specific cell types. In the present work, we attempt to generate viral particles for targeting gene delivery. We have used CCR5-positive cells as the target for our strategy. Therefore, we developed a novel Sindbis pseudotyped lentiviral vector where the Sindbis receptor binding envelope protein was modified to directly encode a single-chain antibody fragment (scFv) against the CCR5 chemokine receptor. We have generated two chimeric scFv-Sindbis envelopes, varying the length of the peptide linker that connects the heavy chain and light chain of anti-CCR5 scFv. The two chimeric scFv-Sindbis envelopes were successfully incorporated into lentiviral-derived vectors, and the resulting pseudotyped viral particles showed specific targeting to CCR5-expressing cells. However, our data demonstrate that the length of the peptide linker significantly affects the efficiency of infection. Pseudotyped viral particles, which display single-chain antibody fragments with longer peptide linkers, allowed higher titers of infection. The present study can be a model strategy for specific gene delivery mediated by lentiviral vectors pseudotyped with Sindbis envelope displaying scFv that recognizes specific cellular surface proteins. Furthermore, this strategy has the potential to become a powerful approach for targeting gene delivery in anti- HIV gene therapy due to the important role of CCR5 expression in disease progression.

Activation of protein kinase Cδ by proteolytic cleavage contributes to manganese-induced apoptosis in dopaminergic cells: protective role of Bcl-2

Chronic inorganic manganese exposure causes selective toxicity to the nigrostriatal dopaminergic system, resulting in a Parkinsonian-like neurological condition known as Manganism. Apoptosis has been shown to occur in manganese-induced neurotoxicity; however, the down-stream cellular target of caspase-3 that contributes to DNA fragmentation is not established. Herein, we demonstrate that proteolytic activation of protein kinase Cδ (PKCδ) by caspase-3 plays a critical role in manganese-induced apoptotic cell death. Treatment of PC12 cells with manganese caused a sequential activation of mitochondrial-dependent pro-apoptotic events, including mitochondrial membrane depolarization, cytochrome c release, caspase-3 activation, and DNA fragmentation. Overexpression of Bcl-2 in PC12 cells remarkably attenuated each of these events, indicating that the mitochondrial-dependent apoptotic cascade contributes to manganese-induced apoptosis. Furthermore, PKCδ was proteolytically cleaved by caspase-3, causing a persistent activation of the kinase. The manganese-induced proteolytic cleavage of PKCδ was significantly blocked by Bcl-2-overexpression. Administration of active recombinant PKCδ induced DNA fragmentation in PC12 cells, suggesting a pro-apoptotic role of PKCδ. Furthermore, expression of catalytically inactive mutant PKCδ K376R via a lentiviral gene delivery system effectively attenuated manganese-induced apoptosis. Together, these results suggest that the mitochondrial-dependent caspase cascade mediates apoptosis via proteolytic activation of PKCδ in manganese-induced neurotoxicity.