Lentivirus-mediated Gene Delivery Research Articles

Loss of RBFOX2 inhibits meiotic initiation in male mice

Meiotic initiation is a critical step in gametogenesis. Recently, some genes required for meiotic initiation have been identified. However, meiosis-initiating factors and the underlying mechanisms are far from being fully understood. We have established a long-term culture system of spermatogonial stem cells (SSCs) and an in vitro model of meiotic initiation using mouse SSCs. Our previous study revealed that the RNA-binding protein RBFOX2 may regulate meiotic initiation, but the role and the mechanism need to be further elucidated. In this study, we constructed RBFOX2 knockdown SSC lines by using lentivirus-mediated gene delivery method, and found that the knockdown SSCs underwent normal self-renewal, mitosis and differentiation. However, they were unable to initiate meiosis when treated with retinoic acid, and they underwent apoptosis. These results indicate that RBFOX2 plays an essential role in meiotic initiation of spermatogonia. This work provides new clues for understanding the functions of RNA-binding proteins in meiotic initiation.

Scinderin suppresses cell proliferation and predicts the poor prognosis of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) remains an intractable disease despite numerous advancements made in the available treatments over recent decades. Therefore, investigation of the underlying pathogenesis of HCC is urgently required. Our previous microarray result showed that SCIN was generally downregulated in 23 paired tumor/normal tissues. Reverse transcription-quantitative PCR, western blotting and immunohistochemistry were performed in the present study in order to detect the expression of scinderin (SCIN). Lentivirus-mediated gene delivery was used in order to produce SCIN-manipulated cell lines. MTT and crystal violet assays were performed in order to investigate cell growth, and fluorescence-activated cell sorting analysis was used in order to determine cell cycle distribution. SCIN was downregulated in HCC samples, and low SCIN expression predicted the poor prognosis of patients with HCC. Notably, SCIN may have the potential to serve as an independent risk factor for overall survival (3-year overall survival rate of 28.6 and 10.3% in high SCIN expression and low SCIN expression groups, respectively) and disease-free survival (3-year recurrence rate of 71.4 and 84.6% in high SCIN expression and low SCIN expression groups, respectively) in HCC. SCIN inhibited HCC cell proliferation both in vitro and in subcutaneous tumor formation assay. Furthermore, SCIN decreased the levels of phosphorylated STAT3, thereby downregulating cyclin A1 levels in HCC cells. The results of the present study demonstrate the tumor suppressive role of SCIN in HCC, providing a candidate strategy to treat this disease.

Silencing APRIL Gene by siRNA Leads to Inhibition of Proliferation and Cell Cycle Arrest in Gastric Cancer Cell Line MGC-803 and SGC-7901

The aim of this study was to explore the effects of silencing a proliferation-inducing ligand on the growth and cell cycle of human gastric cancer cells. MGC-803 and SGC-7901 cells were infected with lenti-a proliferation-inducing ligand si and mRNA expression was analyzed by real-time polymerase chain reaction. Cell viability, colony formation and cell cycle stages were detected by MTT assay, colony forming assay and Cellomics ArrayScan, respectively. Results from real-time polymerase chain reaction indicated that lenti-a proliferation-inducing ligand-si greatly reduced the proliferation-inducing ligand mRNA expression in both cell lines. Cell viability and colony formation ability were significantly hampered. Moreover, cell cycle was arrested in both cell lines, elucidating the mechanism underlying the inhibitory effects of siRNA on cell proliferation. The present study indicated that lentivirus-mediated gene delivery might be a promising strategy in the treatment of gastric cancer.

PTPRU, As A Tumor Suppressor, Inhibits Cancer Stemness By Attenuating Hippo/YAP Signaling Pathway.

BackgroundPTPRU is an important signaling molecule that regulates a variety of cellular processes; however, the role of PTPRU in cancer development has remained elusive. Here, we report that PTPRU serves as a tumor suppressor that inhibits cancer stemness by attenuating Hippo/YAP signaling pathway.MethodsPrimary cancer cells and cell line cells were used in the study. The gene expression data were downloaded from R2 analysis and visualization platform and Kaplan–Meier analysis was performed to study the relationship between survival and PTPRU expression. qRT-PCR and Western blot were employed to study the expression of target genes in tissues and cells. Sphere and colony formation, proliferation, migration activities and the expression of stem cell and EMT markers were employed for characterizing the stemness. Gene manipulation was achieved by lentivirus-mediated gene delivery system. Luciferase reporter gene assay was used to study the transcriptional activity of the promoter, and ChIP-qPCR was employed to study the target binding sequence of the protein. Spearman correlation analysis was performed to study the correlation between two genes. Student’s t-test was used for determination of the significance between two experimental groups.ResultsPTPRU is downregulated in colorectal and gastric cancer tissues and cancer stem cells. High expression of PTPRU predicts poor prognosis. Overexpression of PTPRU attenuates the stemness of gastric cancer stem cells and knockdown of PTRPU improves the maintenance of the stemness of cancer stem cells. Mechanistic analysis showed that PTPRU inhibits Hippo/YAP signaling by suppressing the expression of YAP in a transcriptional level. Overexpression of YAP restored PTPRU-induced inhibited stemness of gastric cancer stem cells.ConclusionPTPRU serves as a tumor suppressor that inhibits the stemness of cancer stem cell by inhibiting Hippo/YAP signaling pathway.

Combining Engineered U1 snRNA and Antisense Oligonucleotides to Improve the Treatment of a BBS1 Splice Site Mutation

Manipulation of pre-mRNA processing is a promising approach toward overcoming disease-causing mutations and treating human diseases. We show that a combined treatment applying two splice-manipulating technologies improves therapeutic efficacies to correct mutation-induced splice defects. Previously, we identified a family affected by retinitis pigmentosa caused by the homozygous BBS1 splice donor site mutation c.479G > A. The mutation leads to both exon 5 skipping and intron 5 retention. We developed a therapeutic approach applying lentivirus-mediated gene delivery of engineered U1 small nuclear RNA (U1), which resulted in increased levels of correctly spliced BBS1. Herein, we show that the therapeutic effect of the engineered U1 efficiently reverted exon skipping but failed to reduce the intron retention. To complement the engineered U1 treatment, we identified four different antisense oligonucleotides (AONs) that block intron 5 retention in BBS1 transcripts. A treatment using engineered U1 in combination with AONs showed the highest therapeutic efficacy and increased the amount of correctly spliced BBS1 transcripts. We did not detect elevated levels of apoptotic cell death in AON-treated cell lines. In conclusion, engineered U1 or AONs provide efficient therapies with complementary effects and can be combined to increase efficacy of therapeutic approaches to correct splice defects.

MicroRNA-7 as a potential therapeutic target for aberrant NF-\u03baB-driven distant metastasis of gastric cancer

BackgroundDysregulated miR-7 and aberrant NF-κB activation were reported in various human cancers. However, the expression profile, clinical relevance and dysregulated mechanism of miR-7 and NF-κB RelA/p65 in human gastric cancers (GC) metastasis remain largely unknown. This study is to investigate the expression profile, clinical relevance and dysregulated mechanism of miR-7 and NF-κB RelA/p65 in GC and to explore the potential therapeutic effect of miR-7 to GC distant metastasis.MethodsTCGA STAD and NCBI GEO database were used to investigate the expression profile of miR-7 and NF-κB RelA/p65 and clinical relevance. Lentivirus-mediated gene delivery was applied to explore the therapeutic effect of miR-7 in GC. Real-time PCR, FACS, IHC, IF, reporter gene assay, IP, pre-miRNA-7 processing and binding assays were performed.ResultsLow miR-7 correlated with high RelA/p65 in GC with a clinical relevance that low miR-7 and high RelA/p65 as prognostic indicators of poor survival outcome of GC patients. Moreover, an impaired pre-miR-7 processing caused by dysregulated Dicer1 expression is associated with downregulated miR-7 in GC cells. Functionally, delivery of miR-7 displays therapeutic effects to GC lung and liver metastasis by alleviating hemangiogenesis, lymphangiogenesis as well as inflammation cells infiltration. Mechanistically, miR-7 suppresses NF-κB transcriptional activity and its downstream metastasis-related molecules Vimentin, ICAM-1, VCAM-1, MMP-2, MMP-9 and VEGF by reducing p65 and p-p65-ser536 expression. Pharmacologic prevention of NF-κB activator LPS obviously restored miR-7-suppressed NF-κB transcriptional activation and significantly reverted miR-7-inhibited cell migration and invasion.ConclusionsOur data suggest loss of miR-7 in GC promotes p65-mediated aberrant NF-κB activation, facilitating GC metastasis and ultimately resulting in the worse clinical outcome. Thus, miR-7 may act as novel prognostic biomarker and potential therapeutic target for aberrant NF-κB-driven GC distant metastasis.

MTOR inhibitors lower an intrinsic barrier to virus infection mediated by IFITM3.

Rapamycin and its derivatives are specific inhibitors of mammalian target of rapamycin (mTOR) kinase and, as a result, are well-established immunosuppressants and antitumorigenic agents. Additionally, this class of drug promotes gene delivery by facilitating lentiviral vector entry into cells, revealing its potential to improve gene therapy efforts. However, the precise mechanism was unknown. Here, we report that mTOR inhibitor treatment results in down-regulation of the IFN-induced transmembrane (IFITM) proteins. IFITM proteins, especially IFITM3, are potent inhibitors of virus-cell fusion and are broadly active against a range of pathogenic viruses. We found that the effect of rapamycin treatment on lentiviral transduction is diminished upon IFITM silencing or knockout in primary and transformed cells, and the extent of transduction enhancement depends on basal expression of IFITM proteins, with a major contribution from IFITM3. The effect of rapamycin treatment on IFITM3 manifests at the level of protein, but not mRNA, and is selective, as many other endosome-associated transmembrane proteins are unaffected. Rapamycin-mediated degradation of IFITM3 requires endosomal trafficking, ubiquitination, endosomal sorting complex required for transport (ESCRT) machinery, and lysosomal acidification. Since IFITM proteins exhibit broad antiviral activity, we show that mTOR inhibition also promotes infection by another IFITM-sensitive virus, Influenza A virus, but not infection by Sendai virus, which is IFITM-resistant. Our results identify the molecular basis by which mTOR inhibitors enhance virus entry into cells and reveal a previously unrecognized immunosuppressive feature of these clinically important drugs. In addition, this study uncovers a functional convergence between the mTOR pathway and IFITM proteins at endolysosomal membranes.

MicroRNA-212 inhibits oligodendrocytes during maturation by down-regulation of differentiation-associated gene expression.

MicroRNA-212 (mir-212) has been reported to regulate neuronal development and functioning. However, its expression and function in glia are not yet known. Here, we demonstrate that the level of microRNA-212 (mir-212) was reduced in spinal cord lesion site at 1week and 1month after a contusive spinal cord injury. In addition to its expression in neurons, mir-212 expression was detected in oligodendrocytes (OLGs) and glial progenitor cells (GPCs) in adult CNS. The addition of antagomir-212 to reduce mir-212 expression enabled to improve the cell process outgrowth of OLGs along with the up-regulation of the genes associated with OLG differentiation and maturation, including OLIG1, SOX10, myelin basic protein (MBP), and proteolipid protein 1 (PLP1). In contrast, these genes were significantly down-regulated by an increased expression of mir-212 in GPCs or in OLG progenitor cells (OPCs) through lentivirus-mediated gene delivery approach. Moreover, we found that PLP1 was the direct target molecule of mir-212. Furthermore, mir-212 over-expression diminished the protein production of OLGs markers including 2',3'-cyclic-nucleotide 3'-phosphodiesterase (CNPase), MBP, and PLP. Additionally, mir-212 over-expression decreased the number of mature OLGs expressing MBP, and the expression of galactocerebroside (GC). Complementary studies in a hippocampal neuron-OLG co-culture model and an exvivo cerebellar slice system indicated that OLGs derived from GPCs with mir-212 over-expression exhibited decreased ability to interact with neuronal axons. Collectively, our findings demonstrate that mir-212 repressed the expression of OLG maturation-associated proteins and inhibited OLG cell process extension, indicating that mir-212 has negative regulatory effect on OLG lineage progression.

Knockdown of PYCR1 inhibits cell proliferation and colony formation via cell cycle arrest and apoptosis in prostate cancer.

Pyrroline-5-carboxylate reductase 1 (PYCR1) is an enzyme involved in cell metabolism, which has been shown to be up-regulated in cancers. However, the functions of PYCR1 in prostate cancers (PCa) are still largely unknown. In the present study, we found that PYCR1 was highly expressed in prostate cancer tissues and then knocked down PYCR1 in PCa cell lines (DU145, PC-3 and LNCap) via lentivirus-mediated gene delivery and analyzed its biological function. Both qRT-PCR and western blotting indicated that PYCR1 was suppressed efficiently after sh-PYCR1 infection. Further analysis indicated knockdown of PYCR1 significantly inhibited PCa cell growth and colony formation ability. The inhibition effects on growth were likely due to G2/M-phase arrest and enhanced cell apoptosis, as determined by flow cytometer analysis. At last, we verified the expression levels of cell cycle regulatory proteins, including CDK1, CDK2, CDK4 and Cyclin B1 were all downregulated and cell apoptotic-related proteins, including cleaved caspase 3 and cleaved PARP were increased in PCa cells after PYCR1 knockdown. Furthermore, PYCR1 has been shown not to be directly regulated by androgen receptor (AR) levels. These results show the functions of PYCR1 in PCa tumorigenesis for the first time and suggest that PYCR1 might be a good potential therapy approach for treating PCa.

Monitoring Cell-autonomous Circadian Clock Rhythms of Gene Expression Using Luciferase Bioluminescence Reporters

In mammals, many aspects of behavior and physiology such as sleep-wake cycles and liver metabolism are regulated by endogenous circadian clocks (reviewed). The circadian time-keeping system is a hierarchical multi-oscillator network, with the central clock located in the suprachiasmatic nucleus (SCN) synchronizing and coordinating extra-SCN and peripheral clocks elsewhere. Individual cells are the functional units for generation and maintenance of circadian rhythms, and these oscillators of different tissue types in the organism share a remarkably similar biochemical negative feedback mechanism. However, due to interactions at the neuronal network level in the SCN and through rhythmic, systemic cues at the organismal level, circadian rhythms at the organismal level are not necessarily cell-autonomous. Compared to traditional studies of locomotor activity in vivo and SCN explants ex vivo, cell-based in vitro assays allow for discovery of cell-autonomous circadian defects. Strategically, cell-based models are more experimentally tractable for phenotypic characterization and rapid discovery of basic clock mechanisms. Because circadian rhythms are dynamic, longitudinal measurements with high temporal resolution are needed to assess clock function. In recent years, real-time bioluminescence recording using firefly luciferase as a reporter has become a common technique for studying circadian rhythms in mammals, as it allows for examination of the persistence and dynamics of molecular rhythms. To monitor cell-autonomous circadian rhythms of gene expression, luciferase reporters can be introduced into cells via transient transfection or stable transduction. Here we describe a stable transduction protocol using lentivirus-mediated gene delivery. The lentiviral vector system is superior to traditional methods such as transient transfection and germline transmission because of its efficiency and versatility: it permits efficient delivery and stable integration into the host genome of both dividing and non-dividing cells. Once a reporter cell line is established, the dynamics of clock function can be examined through bioluminescence recording. We first describe the generation of P(Per2)-dLuc reporter lines, and then present data from this and other circadian reporters. In these assays, 3T3 mouse fibroblasts and U2OS human osteosarcoma cells are used as cellular models. We also discuss various ways of using these clock models in circadian studies. Methods described here can be applied to a great variety of cell types to study the cellular and molecular basis of circadian clocks, and may prove useful in tackling problems in other biological systems.

NF-κB Regulates MICA Gene Transcription in Endothelial Cell through a Genetically Inhibitable Control Site

Endothelial cells form a barrier between blood and the underlying vessel wall, which characteristically demonstrates inflammatory damage in atherosclerotic disease. MICA is a highly polymorphic ligand for the activating immune receptor NKG2D and can be expressed on endothelial cells. We hypothesized that damaged vessel walls, such as those involved in atherosclerosis, might express MICA, which could contribute to the vascular immunopathology. Immune activation resulting from MICA expression could play a significant role in the development of vascular damage. We have demonstrated that TNFα up-regulates MICA on human endothelial cells. The up-regulation is mediated by NF-κB, and we have defined the regulatory control site responsible for this at -130 bp upstream of the MICA transcription start site. This site overlaps with a heat shock response element and integrates input from the two pathways. We have shown that in atherosclerotic lesions there is expression of MICA on endothelial cells. Using lentivirus-mediated gene delivery in primary human endothelial cells, we were able to inhibit the MICA response to TNFα with a truncated HSF1 that lacked a transactivation domain. This highlights the potential for transcription-based therapeutic approaches in atherosclerotic vascular disease to reduce immune-mediated endothelial and vessel wall damage.

Role of Novel Rat-specific Fc Receptor in Macrophage Activation Associated with Crescentic Glomerulonephritis

Crescentic glomerulonephritis (Crgn) is a complex disease where the initial insult is often the glomerular deposition of antibodies against intrinsic or deposited antigens in the glomerulus. The role of Fc receptors in the induction and progression of Crgn is increasingly recognized, and our previous studies have shown that copy number variation in Fcgr3 partially explains the genetic susceptibility of the Wistar-Kyoto (WKY) rat to nephrotoxic nephritis, a rat model of Crgn. The Fcgr3-related sequence (Fcgr3-rs) is a novel rat-specific Fc receptor with a cytoplasmic domain 6 amino acids longer than its paralogue, Fcgr3. The Fcgr3-rs gene is deleted from the WKY rat genome, and this deletion is associated with enhanced macrophage activity in this strain. Here, we investigated the mechanism by which the deletion of Fcgr3-rs in the WKY strain leads to increased macrophage activation. By lentivirus-mediated gene delivery, we generated stably transduced U937 cells expressing either Fcgr3-rs or Fcgr3. In these cells, which lack endogenous Fcgr3 receptors, we show that Fcgr3-rs interacts with the common Fc-γ chain but that Fc receptor-mediated phagocytosis and signaling are defective. Furthermore, in primary macrophages, expression of Fcgr3-rs inhibits Fc receptor-mediated functions, because WKY bone marrow-derived macrophages transduced with Fcgr3-rs had significantly reduced phagocytic activity. This inhibitory effect on phagocytosis was mediated by the novel cytoplasmic domain of Fcgr3-rs. These results suggest that Fcgr3-rs may act to inhibit Fcgr3-mediated signaling and phagocytosis and could be considered as a novel mechanism in the modulation of Fc receptor-mediated cell activation in autoimmune diseases.

Lentiviral-mediated Knockdown During <em>Ex Vivo</em> Erythropoiesis of Human Hematopoietic Stem Cells

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells (HSCs) differentiate into oligopotent progenitors, committed precursors and mature red blood cells. This process is regulated for a large part at the level of gene expression, whereby specific transcription factors activate lineage-specific genes while concomitantly repressing genes that are specific to other cell types. Studies on transcription factors regulating erythropoiesis are often performed using human and murine cell lines that represent, to some extent, erythroid cells at given stages of differentiation. However transformed cell lines can only partially mimic erythroid cells and most importantly they do not allow one to comprehensibly study the dynamic changes that occur as cells progress through many stages towards their final erythroid fate. Therefore, a current challenge remains the development of a protocol to obtain relatively homogenous populations of primary HSCs and erythroid cells at various stages of differentiation in quantities that are sufficient to perform genomics and proteomics experiments. Here we describe an ex vivo cell culture protocol to induce erythroid differentiation from human hematopoietic stem/progenitor cells that have been isolated from either cord blood, bone marrow, or adult peripheral blood mobilized with G-CSF (leukapheresis). This culture system, initially developed by the Douay laboratory, uses cytokines and co-culture on mesenchymal cells to mimic the bone marrow microenvironment. Using this ex vivo differentiation protocol, we observe a strong amplification of erythroid progenitors, an induction of differentiation exclusively towards the erythroid lineage and a complete maturation to the stage of enucleated red blood cells. Thus, this system provides an opportunity to study the molecular mechanism of transcriptional regulation as hematopoietic stem cells progress along the erythroid lineage. Studying erythropoiesis at the transcriptional level also requires the ability to over-express or knockdown specific factors in primary erythroid cells. For this purpose, we use a lentivirus-mediated gene delivery system that allows for the efficient infection of both dividing and non-dividing cells. Here we show that we are able to efficiently knockdown the transcription factor TAL1 in primary human erythroid cells. In addition, GFP expression demonstrates an efficiency of lentiviral infection close to 90%. Thus, our protocol provides a highly useful system for characterization of the regulatory network of transcription factors that control erythropoiesis.

Lentiviral-mediated Knockdown During <em>Ex Vivo</em> Erythropoiesis of Human Hematopoietic Stem Cells

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells (HSCs) differentiate into oligopotent progenitors, committed precursors and mature red blood cells 1. This process is regulated for a large part at the level of gene expression, whereby specific transcription factors activate lineage-specific genes while concomitantly repressing genes that are specific to other cell types 2. Studies on transcription factors regulating erythropoiesis are often performed using human and murine cell lines that represent, to some extent, erythroid cells at given stages of differentiation 3-5. However transformed cell lines can only partially mimic erythroid cells and most importantly they do not allow one to comprehensibly study the dynamic changes that occur as cells progress through many stages towards their final erythroid fate. Therefore, a current challenge remains the development of a protocol to obtain relatively homogenous populations of primary HSCs and erythroid cells at various stages of differentiation in quantities that are sufficient to perform genomics and proteomics experiments. Here we describe an ex vivo cell culture protocol to induce erythroid differentiation from human hematopoietic stem/progenitor cells that have been isolated from either cord blood, bone marrow, or adult peripheral blood mobilized with G-CSF (leukapheresis). This culture system, initially developed by the Douay laboratory 6, uses cytokines and co-culture on mesenchymal cells to mimic the bone marrow microenvironment. Using this ex vivo differentiation protocol, we observe a strong amplification of erythroid progenitors, an induction of differentiation exclusively towards the erythroid lineage and a complete maturation to the stage of enucleated red blood cells. Thus, this system provides an opportunity to study the molecular mechanism of transcriptional regulation as hematopoietic stem cells progress along the erythroid lineage. Studying erythropoiesis at the transcriptional level also requires the ability to over-express or knockdown specific factors in primary erythroid cells. For this purpose, we use a lentivirus-mediated gene delivery system that allows for the efficient infection of both dividing and non-dividing cells 7. Here we show that we are able to efficiently knockdown the transcription factor TAL1 in primary human erythroid cells. In addition, GFP expression demonstrates an efficiency of lentiviral infection close to 90%. Thus, our protocol provides a highly useful system for characterization of the regulatory network of transcription factors that control erythropoiesis.

Activation of Transgene-specific T Cells Following Lentivirus-mediated Gene Delivery to Mouse Lung

Integrating lentiviral vectors based on the human immunodeficiency virus type-1 (HIV-1) can transduce quiescent cells, which in lung account for almost 95% of the epithelial cell population. Pseudotyping lentiviral vectors with the envelope glycoprotein from the Ebola Zaire virus, the lymphocytic choriomeningitis virus (LCMV), the Mokola virus, and the vesicular stomatitis virus (VSV-G) resulted in transduction of mouse alveolar epithelium, but gene expression in the lung of C57BL/6 and BALB/c mice waned within 90 days of vector injection. Intratracheal delivery of the four pseudotyped lentiviral vectors resulted in transgene-specific T-cell activation in both mouse strains, albeit lower than that achieved by intramuscular injection of the vectors. We performed an adoptive transfer of luciferase-specific T cells, isolated from spleen or lung of donor mice injected with VSV-G-pseudotyped lentivirus vector expressing luciferase into the muscle or lung, respectively, into recipient recombination-activating gene (RAG)–deficient mice transduced in lung with adenovirus expressing firefly luciferase (ffluc2). Gene expression declined within 7 days of adoptive transfer approaching background levels by day 36. Taken together, our results suggest that the loss of transduced cells in lung is due to VSV-G.HIV vector–mediated activation of transgene-specific T cells rather than as result of normal turnover of airway cells.

(Strept)avidin-displaying lentiviruses as versatile tools for targeting and dual imaging of gene delivery

Lentiviruses have shown great promise for human gene therapy. However, no optimal strategies are yet available for noninvasive imaging of virus biodistribution and subsequent transduction in vivo. We have developed a dual-imaging strategy based on avidin-biotin system allowing easy exchange of the surface ligand on HIV-derived lentivirus envelope. This was achieved by displaying avidin or streptavidin fused to the transmembrane anchor of vesicular stomatitis virus G protein on gp64-pseudotyped envelopes. Avidin and streptavidin were efficiently incorporated on virus particles, which consequently showed binding to biotin in ELISA. These vectors, conjugated to biotinylated radionuclides and engineered to express a ferritin transgene, enabled for the first-time dual imaging of virus biodistribution and transduction pattern by single-photon emission computed tomography and magnetic resonance imaging after stereotactic injection into rat brain. In addition, vector retargeting to cancer cells overexpressing CD46, epidermal growth factor and transferrin receptors using biotinylated ligands and antibodies was demonstrated in vitro. In conclusion, we have generated novel lentivirus vectors for noninvasive imaging and targeting of lentivirus-mediated gene delivery. This study suggests that these novel vectors could be applicable for the treatment of central nervous system disorders and cancer.

Retronectin enhances lentivirus-mediated gene delivery into hematopoietic progenitor cells

Genetic modification of hematopoietic stem cells holds great promise in the treatment of hematopoietic disorders. However, clinical application of gene delivery has been limited, in part, by low gene transfer efficiency. To overcome this problem, we investigated the effect of retronectin (RN) on lentiviral-mediated gene delivery into hematopoietic progenitor cells (HPCs) derived from bone marrow both in vitro and in vivo. RN has been shown to enhance transduction by promoting colocalization of lentivirus and target cells. We found that RN enhanced lentiviral transfer of the VENUS transgene into cultured c-Kit + Lin − HPCs. As a complementary approach, in vivo gene delivery was performed by subjecting mice to intra-bone marrow injection of lentivirus or a mixture of RN and lentivirus. We found that co-injection with RN increased the number of VENUS-expressing c-Kit + Lin − HPCs in bone marrow by 2-fold. Further analysis of VENUS expression in colony-forming cells from the bone marrow of these animals revealed that RN increased gene delivery among these cells by 4-fold. In conclusion, RN is effective in enhancing lentivirus-mediated gene delivery into HPCs.

Etk/Bmx mediates expression of stress-induced adaptive genes VEGF, PAI-1, and iNOS via multiple signaling cascades in different cell systems

We recently showed that Etk/Bmx, a member of the Tec family of nonreceptor protein tyrosine kinases, promotes tight junction formation during chronic hypoxic exposure and augments normoxic VEGF expression via a feedforward mechanism. Here we further characterized Etk's role in potentiating hypoxia-induced gene expression in salivary epithelial Pa-4 cells. Using transient transfection in conditionally activated Etk (DeltaEtk:ER) cells, we demonstrated that Etk enhances hypoxia-response element-dependent reporter activation in normoxia and hypoxia. This Etk-driven reporter activation is ameliorated by treatment with wortmannin or LFM-A13. Using lentivirus-mediated gene delivery and small interfering RNA, we provided direct evidence that hypoxia leads to transient Etk and Akt activation and hypoxia-mediated Akt activation is Etk dependent. Northern blot analyses confirmed that Etk activation led to induction of steady-state mRNA levels of endogenous VEGF and plasminogen activator inhibitor (PAI)-1, a hallmark of hypoxia-mediated gene regulation. We also demonstrated that Etk utilizes a phosphatidylinositol 3-kinase/Akt pathway to promote reporter activation driven by NF-kappaB, another oxygen-sensitive transcription factor, and to augment cytokine-induced inducible nitric oxide synthase expression in endothelial cells. To establish the clinical relevance of Etk-induced, hypoxia-mediated gene regulation, we examined Etk expression in keloid, which has elevated VEGF and PAI-1. We found that Etk is overexpressed in keloid (but not normal skin) tissues. The differential steady-state Etk protein levels were further confirmed in primary fibroblast cultures derived from these tissues, suggesting an Etk role in tissue fibrosis. Our results provide further understanding of Etk function within multiple signaling cascades to govern adaptive cytoprotection against extracellular stress in different cell systems, salivary epithelial cells, brain endothelial cells, and dermal fibroblasts.

Transduction of human islets with the lentiviral vector

Establishment of an efficient gene delivery system for human pancreatic beta cells is important for the development of diabetes-targeted cell therapies. The human immunodeficiency virus type 1 (HIV-1)–derived lentiviral vector is well documented to be an effective gene transfer tool for various types of cells. Thus, we examined the efficiency of lentivirus-mediated gene delivery for human islets. Human islets were isolated using defined protocols for enzymatic dissociation and purification using discontinuous Ficoll gradients with a refrigerated Cobe 2991 machine. Isolated islets were shipped to Japan, cryopreserved for 3 months, and then subjected to transduction. A vesicular stomatitis virus G-protein (VSV-G)-pseudotyped lentiviral vector LtV-NLS/LacZ was produced in 293T cells under the Fugene6 method. 804G extracellular matrices were applied for monolayer formation of islets. Detection of NLS/LacZ expression was performed using X-gal staining. Lentiviral transduction was effective in these monolayer islets.

Osteosarcoma and chondrosarcoma as targets for virus vectors and herpes simplex virus thymidine kinase/ganciclovir gene therapy

Osteosarcoma and chondrosarcoma, the most prevalent primary malignant tumors of the bone, have been demonstrated to be potential target diseases for herpes simplex virus type 1 thymidine kinase (HSV-TK)/ganciclovir (GCV) suicide gene therapy. However, the utility of this gene therapy form for bone tumor cells has not been studied systematically. In this report we show, with the aid of three osteosarcoma cell lines (Saos-2, U-2-OS and MG-63) and one chondrosarcoma cell line (SW1353) that: i) these tumor cells were permissive for adenovirus- or lentivirus-mediated gene delivery; ii) the cell lines appeared to be good or excellent targets for HSV-TK/GCV gene therapy; and iii) the extent of HSV-TK/GCV cytotoxic effect correlated with the presence of the 'bystander effect' in these cells. Our results also suggest that lentiviruses are potential vectors for bone cancer gene therapy. They transduced all four cell lines with high efficiency and provided HSV-TK expression level that was sufficient for cytotoxicity and bystander effect comparable to that obtained with adenovirus vectors.