Lentivirus-based Gene Research Articles

Anti-Apoptotic Effects of Lentiviral Vector Transduction Promote Increased Rituximab Tolerance in Cancerous B-Cells.

Diffuse large B-cell lymphoma (DLBCL) is characterized by great genetic and clinical heterogeneity which complicates prognostic prediction and influences treatment efficacy. The most common regimen, R-CHOP, consists of a combination of anthracycline- and immuno-based drugs including Rituximab. It remains elusive how and to which extent genetic variability impacts the response and potential tolerance to R-CHOP. Hence, an improved understanding of mechanisms leading to drug tolerance in B-cells is crucial, and modelling by genetic intervention directly in B-cells is fundamental in such investigations. Lentivirus-based gene vectors are widely used gene vehicles, which in B-cells are an attractive alternative to potentially toxic transfection-based methodologies. Here, we investigate the use of VSV-G-pseudotyped lentiviral vectors in B-cells for exploring the impact of microRNAs on tolerance to Rituximab. Notably, we find that robust lentiviral transduction of cancerous B-cell lines markedly and specifically enhances the resistance of transduced germinal center B-cells (GCBs) to Rituximab. Although Rituximab works partially through complement-mediated cell lysis, increased tolerance is not achieved through effects of lentiviral transduction on cell death mediated by complement. Rather, reduced levels of PARP1 and persistent high levels of CD43 in Rituximab-treated GCBs demonstrate anti-apoptotic effects of lentiviral transduction that may interfere with the outcome and interpretation of Rituximab tolerance studies. Our findings stress that caution should be exercised exploiting lentiviral vectors in studies of tolerance to therapeutics in DLBCL. Importantly, however, we demonstrate the feasibility of using the lentiviral gene delivery platform in studies addressing the impact of specific microRNAs on Rituximab responsiveness.

Human, Pig, and Mouse Interferon-Induced Transmembrane Proteins Partially Restrict Pseudotyped Lentiviral Vectors.

Lentiviral vectors are increasingly used in clinical trials to treat genetic diseases. Our research has focused on strategies to improve lentiviral gene transfer efficiency in the airways. Previously we demonstrated that a feline immunodeficiency virus (FIV)-based lentiviral vector pseudotyped with the baculovirus envelope glycoprotein GP64 (GP64-FIV) efficiently transduced mouse nasal epithelia in vivo but transduced mouse intrapulmonary airways with 10-fold less efficiency. Here, we demonstrate that members of a family of proteins with antiviral activity, interferon-induced transmembrane proteins (IFITMs), are more highly expressed in mouse intrapulmonary airways as compared with mouse nasal airways. Using GP64- and VSV-G (vesicular stomatitis virus G glycoprotein)-pseudotyped FIV, we show that expression of mouse IFITM1, IFITM2, and IFITM3 restricts gene transfer. Further, we show that both the nasal and intrapulmonary airways of IFITM locus knockout mice are more efficiently transduced with GP64-FIV than their heterozygous littermates. In anticipation of transitioning our studies into pig models of airway disease and clinical trials in humans, we investigated the ability of pig and human IFITMs to restrict lentiviral gene transfer. We observed that both human and pig IFITMs partially restricted both VSV-G-FIV and GP64-FIV transduction in vitro. Previous studies have focused on IFITM-mediated restriction of replication-competent wild-type viruses; however, these results implicate the IFITM proteins as restriction factors that can limit lentivirus-based vector gene transfer to airway epithelia. The findings are relevant to future preclinical and clinical airway gene therapy trials using lentivirus-based vectors.

Lentivirus-Based Stable Gene Delivery into Intestinal Organoids.

Lentivirus-based gene delivery works efficiently for the majority of mammalian cells cultured under standard two-dimensional conditions. By contrast, intestinal epithelial organoids embedded into three-dimensional extracellular matrix appear to be resistant to lentiviral transduction. We observed that Matrigel, a matrix that reconstitutes a basement membrane and is indispensable for cell survival and proliferation, prevents lentiviruses from binding to intestinal cells. In this chapter, we describe a simple method of a highly efficient gene transduction into intestinal organoids. This method involves organoid dispersion into single intestinal epithelial cells, mixing these individual cells with lentiviral particles, plating on Matrigel, and subsequent re-embedding into Matrigel. Under these conditions, the majority of the cells are exposed to the virus in the absence of the matrix barrier while remaining attached to the matrix. Using a GFP-labeled lentivirus, we demonstrate that this method allows for highly efficient infection of intestinal organoids after overnight incubation of Matrigel-attached cells with lentiviral particles.

Double transduction of a Cre/LoxP lentiviral vector: a simple method to generate kidney cell-specific knockdown mice.

In a lentivirus-based gene delivery system, the incorporated gene is continuously expressed for a long time. In this study, we devised a simple way to knock down a specific gene in a kidney cell-specific pattern in adult mice by lentivirus-assisted transfer of short hairpin RNA (shRNA). Kidney collecting duct (CD)-specific aquaporin-3 (AQP3)-knockdown mice were generated by consecutive injection of Hoxb7-Cre-expressing lentivirus (LV-Hoxb7 Cre) and loxP-AQP3 shRNA-expressing lentivirus (LV-loxP shAQP3) in adult C57BL6/J mice. LV-Hoxb7 Cre was designed to express mCherry, while LV-loxP shAQP3 was designed with a floxed enhanced green fluorescent protein (EGFP)-tagged stop sequence, and thus EGFP would be expressed only in the absence of Cre recombination. In mice treated with LV-Hoxb7 Cre alone, mCherry protein expression, which indicates the presence of Cre recombinase, occurred only in CD cells. However, LV-loxP shAQP3 injection alone resulted in an increase in EGFP expression in all kidney cells, indicating the transcription of the floxed region. When LV-Hoxb7 Cre and LV-loxP shAQP3 were sequentially transduced, EGFP expression was attenuated while mCherry expression was sustained in CD cells, demonstrating a CD cell-specific recombination of the floxed region. AQP3 expression in mice injected with LV-Hoxb7 Cre or LV-loxP shAQP3 alone did not differ, but consecutive injection of LV-Hoxb7 Cre and LV-loxP shAQP3 significantly reduced AQP3 expression in CD cells. However, the expression levels of AQP3 were not altered in other cell types. Double transduction of Cre- and loxP-based lentivirus can easily generate kidney cell-specific knockdown mice, and this method might be applicable to other species.

Vpx mediated degradation of SAMHD1 has only a very limited effect on lentiviral transduction rate in ex vivo cultured HSPCs

Understanding how to achieve efficient transduction of hematopoietic stem and progenitor cells (HSPCs), while preserving their long-term ability to self-reproduce, is key for applying lentiviral-based gene engineering methods. SAMHD1 is an HIV-1 restriction factor in myeloid and resting CD4+ T cells that interferes with reverse transcription by decreasing the nucleotide pools or by its RNase activity. Here we show that SAMHD1 is expressed at high levels in HSPCs cultured in a medium enriched with cytokines. Thus, we hypothesized that degrading SAMHD1 in HSPCs would result in more efficient lentiviral transduction rates. We used viral like particles (VLPs) containing Vpx, shRNA against SAMHD1, or provided an excess of dNTPs or dNs to study this question. Regardless of the method applied, we saw no increase in the lentiviral transduction rate. The result was different when we used viruses (HR-GFP-Vpx+) which carry Vpx and encode GFP. These viruses allow assessment of the effects of Vpx specifically in the transduced cells. Using HR-GFP-Vpx+ viruses, we observed a modest but significant increase in the transduction efficiency. These data suggest that SAMHD1 has some limited efficacy in blocking reverse transcription but the major barrier for efficient lentiviral transduction occurs before reverse transcription.

Effects of Notch2 and Notch3 on Cell Proliferation and Apoptosis of Trophoblast Cell Lines.

Aims: To investigate the effect of Notch2 and Notch3 on cell proliferation and apoptosis of two trophoblast cell lines, BeWo and JAR.Methods: Notch2 and Notch3 expression in BeWo and JAR cells was upregulated or downregulated using lentivirus-mediated overexpression or RNA interference. The effect of Notch2 and Notch3 on cell proliferation was assessed by the CCK-8 assay. The effect of Notch2 and Notch3 on the apoptosis of BeWo and JAR cells was evaluated by flow cytometry using the Annexin V-PE Apoptosis kit. Lentivirus-based overexpression vectors were constructed by cloning the full-length coding sequences of human Notch2 and Notch3 C-terminally tagged with GFP or GFP alone (control) into a lentivirus-based expression vector. Lentivirus-based gene silencing vectors were prepared by cloning small interfering sequences targeting human Notch2 and Notch3 and scrambled control RNA sequence into a lentivirus-based gene knockdown vector. The effect of Notch2 and Notch3 on cell proliferation was assessed by the CCK-8 assay. And the effect of Notch2 and Notch3 on the apoptosis of BeWo and JAR cells was evaluated by flow cytometry using the Annexin V PE Apoptosis kit.Results: We found that the downregulation of Notch2 and Notch3 gene expression in BeWo and JAR cells resulted in an increase in cell proliferation, while upregulation of Notch3 and Notch2 expression led to a decrease in cell proliferation. Moreover, the overexpression of Notch3 and Notch2 in BeWo and JAR cells reduced apoptosis in these trophoblast cell lines, whereas apoptosis was increased in the cells in which the expression of Notch3 and Notch2 was downregulated.Conclusions: Notch2 and Notch3 inhibited both cell proliferation and cell apoptosis in BeWo and JAR trophoblast cell lines.

Multigenic lentiviral vectors for combined and tissue-specific expression of miRNA- and protein-based antiangiogenic factors

Lentivirus-based gene delivery vectors carrying multiple gene cassettes are powerful tools in gene transfer studies and gene therapy, allowing coexpression of multiple therapeutic factors and, if desired, fluorescent reporters. Current strategies to express transgenes and microRNA (miRNA) clusters from a single vector have certain limitations that affect transgene expression levels and/or vector titers. In this study, we describe a novel vector design that facilitates combined expression of therapeutic RNA- and protein-based antiangiogenic factors as well as a fluorescent reporter from back-to-back RNApolII-driven expression cassettes. This configuration allows effective production of intron-embedded miRNAs that are released upon transduction of target cells. Exploiting such multigenic lentiviral vectors, we demonstrate robust miRNA-directed downregulation of vascular endothelial growth factor (VEGF) expression, leading to reduced angiogenesis, and parallel impairment of angiogenic pathways by codelivering the gene encoding pigment epithelium-derived factor (PEDF). Notably, subretinal injections of lentiviral vectors reveal efficient retinal pigment epithelium-specific gene expression driven by the VMD2 promoter, verifying that multigenic lentiviral vectors can be produced with high titers sufficient for in vivo applications. Altogether, our results suggest the potential applicability of combined miRNA- and protein-encoding lentiviral vectors in antiangiogenic gene therapy, including new combination therapies for amelioration of age-related macular degeneration.

Waking the Sleeping Giant: Gene Therapy in Decline?

A search of the literature shows that overall interest in gene therapy, as indicated by publication numbers, has been on a slow decline for over a decade, since the year 2000. In spite of this the stunning potential of gene therapy to revolutionize modern medicine remains unfulfilled. The bottleneck of NIH funding for basic research and lack of means to enter into the clinical trials pipeline (toxicology and Phase I-III clinical trials) strongly limits advancements in gene therapy-based medical breakthroughs. Within the declining general field, publications on adeno-associated virus (AAV) - and lentivirus-based gene delivery continue to rise and offer some hope for the future.

Differentiation of Human-Induced Pluripotent Stem Cells (iPSCs) on Laminin-functionalized PEG Hydrogels under Nucleus Pulposus-Like Culture Conditions

IntroductionCell delivery to the pathological intervertebral disc (IVD) has significant therapeutic potential for enhancing IVD regeneration1-4; however, few viable sources of cells have been ident...

Robust Expression of the Human Neonatal Fc Receptor in a Truncated Soluble Form and as a Full-Length Membrane-Bound Protein in Fusion with eGFP

Studies on the neonatal Fc receptor (FcRn) have revealed a multitude of important functions in mammals, including protection of IgG and serum albumin (SA) from lysosomal degradation. The pharmacokinetic behavior of therapeutic antibodies, IgG-Fc- and SA-containing drugs is therefore influenced by their interaction with FcRn. Pre-clinical development of such drugs is facilitated if their interaction with FcRn can be studied in vitro. For this reason we have developed a robust system for production of the soluble extracellular domain of human FcRn as well as the full-length receptor as fusion to green fluorescent protein, taking advantage of a lentivirus-based gene delivery system where stable over-expressing cells are easily and rapidly generated. Production of the extracellular domain in multiple-layered culture flasks, followed by affinity purification using immobilized IgG, resulted in capture of milligram amounts of soluble receptor per liter cell culture with retained IgG binding. The receptor was further characterized by SDS-PAGE, western blotting, circular dichroism spectroscopy, ELISA, surface plasmon resonance and a temperature stability assay showing a functional and stable protein of high purity. The full-length receptor was found to be successfully over-expressed in a membrane-bound form with retained pH-dependent IgG- and SA-binding.

Culture Of Mobilized Rhesus Macaque CD34+ Cells In Hypoxic Conditions Does Not Improve Lentiviral Transduction Efficiency In Long-Term Repopulating Hematopoietic Stem Cells

Low oxygen levels have been proposed to play a fundamental role in the maintenance of normal hematopoietic stem cells (HSC) function. We investigated whether culture of rhesus macaque mobilized CD34+ cells under low oxygen conditions (1-5% O2) could improve lentiviral transduction efficiency in HSCs compared to culture under atmospheric O2 conditions (21%). Mobilized CD34+ cells from 4 animals were prestimulated for 12 hours in the presence of cytokines and subsequently divided in two equal fractions for transduction with SIN-lentiviral vectors (MOI=100) expressing GFP or TdTomato fluorescent proteins for 48 hours. In 3 animals, cells were transduced with GFP vectors in hypoxia and TdTomato vectors in normoxia. These conditions were reversed in one animal. A portion of the transduced cells was used for phenotypic, cell cycle, clonogenic and apoptosis assays. The remaining cells from both fractions were combined and injected intravenously in lethally irradiated rhesus macaques and in vivo gene marking was measured in peripheral blood granulocytes for up to 21 months after transplantation. The numbers of total live cells and CD34+ cells after 2.5 days in culture were not significantly different compared to baseline for both hypoxic and normoxic conditions (p=0.85 and p=0.84, respectively). Similarly, numbers of CD34+CD38- cells were stable compared to baseline in hypoxia and normoxia (p=0.94). After 2.5 days of culture, the total number of colony-forming cells increased 1.4-fold under both hypoxic and normoxic conditions (p=0.69) compared to freshly isolated CD34+ cells. At baseline, the majority (50.6 + 4.4%) of the CD34+ cells were in the G0 phase of the cell cycle. After 2.5 days in culture under hypoxic or normoxic conditions, the percentages of cells in G0 were 19.9 + 8.8% and 18.6 + 5.9%, respectively (p=0.82). The differences in percentages of cells in the G1 and S/G2/M phases of the cell cycle were also not statistically significant (p=0.175 and p=0.732, respectively). The pO2 had no impact on cell death (18.2 + 5.3% in hypoxia and 16.8 + 4.8% in normoxia, p=0.69) or on the percentages of apoptotic cells (11.1 + 6.7% in hypoxia and 8.4 + 2.3% in normoxia, p=0.47). When considering the bulk of CD34+ cells after transduction with lentiviral vectors, the percentages of GFP-marked cells were consistently higher (range 1.8 to 2.2-fold, mean 2.1-fold) compared to TdTomato-marked cells independently of the transduction conditions used, consistent with a 2.1-fold intrinsic superiority of GFP-based lentiviral vectors compared to TdTomato vectors at equivalent MOI. Taking into account the inherent vector differences, transduction efficiencies were similar in normoxia (70.7 + 11.7%) and hypoxia (68.0 + 16.3%) (p=0.80). When adjusted for the intrinsic superiority of GFP-based vectors, the contribution to long-term in vivo gene marking measured by flow cytometry and quantitative PCR was comparable between cells transduced in 21% pO2 (9.0 + 4.5%) and 5% pO2 conditions (8.4 + 4.9%) (p=0.87). Consistent with these data, GFP and TdTomato-labeled hematopoietic cells were equally visualized, using a confocal/2-photon hybrid microscopy approach, in BM biopsy specimens collected at various time points after transplantation. Given recent evidence suggesting that lower oxygen concentrations (<1.5%) may be required to stabilize HIF-1a, a key sensor of hypoxic conditions, transduction was performed under extreme hypoxic conditions (1% O2) in one animal. While the overall viability of the bulk CD34+ cells after 2.5 days of transduction under extreme hypoxia was comparable to cells cultured in normoxia, their contribution to long-term in vivo marking was negligible (0.3%) compared to cells transduced under normoxic conditions (2.4%), suggesting toxicity of very low oxygen levels on HSCs. Overall, when current methodologies used for the genetic manipulation of HSCs for gene therapy applications were performed under hypoxic conditions, susceptibility to lentiviral transduction of CD34+ cells was not ameliorated compared to cells cultured in 21% O2 conditions in the rhesus macaque transplantation model. The short culture times (2-3 days) used in recent lentivirus-based gene therapy clinical trials and replicated in this study may not be sufficient to impact the phenotype of long-term repopulating HSCs and do not warrant incorporation of hypoxia in current gene therapy protocols. Disclosures:No relevant conflicts of interest to declare.

Characterization of Lentiviral Vector Production Using Microwell Suspension Cultures of HEK293T-Derived Producer Cells

ProSavin(®) is a lentiviral vector (LV)-based gene therapy for Parkinson's disease. ProSavin(®) is currently in a Phase I/II clinical trial using material that was generated by transient transfection of adherent human embryonic kidney (HEK)293T cells. For future large-scale productions of ProSavin(®), we have previously reported the development and characterization of two inducible producer cell lines, termed PS5.8 and PS46.2. PS46.2 has been successfully adapted to grow in suspension cultures. The present study describes the creation of a small-scale (<2 ml) microwell-based experimental platform for the parallel investigation of ProSavin(®) production using suspension-adapted PS46.2. This is combined with statistical design of experiments (DoE) techniques to enable rapid characterization of the process conditions that impact cell growth and LV production. The effects of postinduction period, microwell liquid fill volume, and concentration of inducer (doxycycline) on ProSavin(®) titer and the particle:infectivity (P:I) ratio was investigated using three rounds of DoE, in order to identify appropriate factor ranges and optimize production conditions. We identified an optimal "harvest window" between approximately 26-46 hr within which maximal titers of around 6×10(4) transducing units (TU)/ml were obtained (an approximately 30-fold improvement compared to starting microwell conditions), providing that the fill volume was maintained at or below 1 ml and the doxycycline concentration was at least 1.0 μg/ml. Insights from the microwell studies were subsequently used to rapidly establish operating conditions for ProSavin(®) production in a 0.5-L wave bioreactor culture. The information presented herein thus aids the design and evaluation of scalable production processes for LVs.

Simvastatin Suppresses Osteoblastic Expression of Cyr61 and Progression of Apical Periodontitis through Enhancement of the Transcription Factor Forkhead/Winged Helix Box Protein O3a

IntroductionIn this study, the role of transcription factor Forkhead/winged helix box protein O3a (FoxO3a) in Cyr61 expression and its modulation by simvastatin were investigated in cultured murine osteoblasts and a rat model of induced apical periodontitis. We also examined the effects of simvastatin on the synthesis of chemokine CCL2 and chemotaxis of macrophages in vitro. MethodsWe assessed tumor necrosis factor (TNF)-α–stimulated expression of Cyr61 and phosphorylated inactive FoxO3a (p-FoxO3a) in MC3T3-E1 murine osteoblasts by Western analysis. Forced expression of FoxO3a by lentiviral-based gene transduction was performed, and its effect on Cyr61 expression was evaluated. The modulation of CCL2 secretion and macrophage chemotaxis by simvastatin were examined by enzyme-linked immunosorbent assay and transwell migration assay, respectively. In a rat model of induced apical periodontitis, the relation between disease progression and osteoblastic expression of Cyr61, p-FoxO3a, and CCL2 and macrophage recruitment were studied by radiographic and immunohistochemistry analyses. ResultsWestern blot analysis showed enhanced expression of Cyr61 and p-FoxO3a after TNF-α treatment in a time-dependent manner. Simvastatin significantly counteracted the actions of TNF-α. Forced expression of FoxO3a reduced TNF-α–stimulated Cyr61 synthesis. Simvastatin and FoxO3a diminished TNF-α–induced CCL2 secretion and macrophage recruitment, whereas Cyr61 partially restored the stimulating action. In rat periapical lesions, simvastatin significantly attenuated bone resorption, reduced osteoblastic expressions of Cyr61, p-FoxO3a, and CCL2, and suppressed macrophage recruitment. ConclusionsSimvastatin may alleviate periapical lesions by enhancing FoxO3a activity to suppress the synthesis of Cyr61 in osteoblasts. Moreover, the downstream effector mechanism of Cyr61 may involve CCL2 production and macrophage recruitment.

VGLL3 expression is associated with a tumor suppressor phenotype in epithelial ovarian cancer

Previous studies have implicated vestigial like 3 (VGLL3), a chromosome 3p12.3 gene that encodes a putative transcription co-factor, as a candidate tumor suppressor gene (TSG) in high-grade serous ovarian carcinomas (HGSC), the most common type of epithelial ovarian cancer. A complementation analysis based on microcell-mediated chromosome transfer (MMCT) using a centric fragment of chromosome 3 (der3p12-q12.1) into the OV-90 ovarian cancer cell line haploinsufficient for 3p and lacking VGLL3 expression was performed to assess the effect on tumorigenic potential and growth characteristics. Genetic characterization of the derived MMCT hybrids revealed that only the hybrid that contained an intact VGLL3 locus exhibited alterations of tumorigenic potential in a nude mouse xenograft model and various in vitro growth characteristics. Only stable OV-90 transfectant clones expressing low levels of VGLL3 were derived. These clones exhibited an altered cytoplasmic morphology characterized by numerous single membrane bound multivesicular-bodies (MVB) that were not attributed to autophagy. Overexpression of VGLL3 in OV-90 was achieved using a lentivirus-based tetracycline inducible gene expression system, which also resulted in MVB formation in the infected cell population. Though there was no significant differences in various in vitro and in vivo growth characteristics in a comparison of VGLL3-expressing clones with empty vector transfectant controls, loss of VGLL3 expression was observed in tumors derived from mouse xenograft models. VGLL3 gene and protein expression was significantly reduced in HGSC samples (>98%, p < 0.05) relative to either normal ovarian surface epithelial cells or epithelial cells of the fallopian tube, possible tissues of origin of HGSC. Also, there appeared to be to be more cases with higher staining levels in stromal tissue component from HGSC cases that had a prolonged disease-free survival. The results taken together suggest that VGLL3 is involved in tumor suppressor pathways, a feature that is characterized by the absence of VGLL3 expression in HGSC samples.

Antigen-Specific Gene Therapy after Immunisation Reduces the Severity of Collagen-Induced Arthritis

Reestablishment of tolerance induction in rheumatoid arthritis (RA) would be an optimal treatment with few, if any, side effects. However, to develop such a treatment further insights in the immunological mechanisms governing tolerance are needed. We have developed a model of antigen-specific tolerance in collagen type II (CII) induced arthritis (CIA) using lentivirus-based gene therapy. The immunodominant epitope of CII was inserted into a lentivirus vector to achieve expression on the MHC class II molecule and the lentiviral particles were subsequently intravenously injected at different time points during CIA. Injection of lentiviral particles in early phases of CIA, that is, at day 7 or day 26 after CII immunisation, partially prevented development of arthritis, decreased the serum levels of CII-specific IgG antibodies, and enhanced the suppressive function of CII-specific T regulatory cells. When lentiviral particles were injected during manifest arthritis, that is, at day 31 after CII immunisation, the severity of arthritis progression was ameliorated, the levels of CII-specific IgG antibodies decreased and the proportion of T regulatory cells increased. Thus, antigen-specific gene therapy is effective when administered throughout the inflammatory course of arthritis and offers a good model for investigation of the basic mechanisms during tolerance in CIA.

Transient, Inducible, Placenta-Specific Gene Expression in Mice

Molecular understanding of placental functions and pregnancy disorders is limited by the absence of methods for placenta-specific gene manipulation. Although persistent placenta-specific gene expression has been achieved by lentivirus-based gene delivery methods, developmentally and physiologically important placental genes have highly stage-specific functions, requiring controllable, transient expression systems for functional analysis. Here, we describe an inducible, placenta-specific gene expression system that enables high-level, transient transgene expression and monitoring of gene expression by live bioluminescence imaging in mouse placenta at different stages of pregnancy. We used the third generation tetracycline-responsive tranactivator protein Tet-On 3G, with 10- to 100-fold increased sensitivity to doxycycline (Dox) compared with previous versions, enabling unusually sensitive on-off control of gene expression in vivo. Transgenic mice expressing Tet-On 3G were created using a new integrase-based, site-specific approach, yielding high-level transgene expression driven by a ubiquitous promoter. Blastocysts from these mice were transduced with the Tet-On 3G-response element promoter-driving firefly luciferase using lentivirus-mediated placenta-specific gene delivery and transferred into wild-type pseudopregnant recipients for placenta-specific, Dox-inducible gene expression. Systemic Dox administration at various time points during pregnancy led to transient, placenta-specific firefly luciferase expression as early as d 5 of pregnancy in a Dox dose-dependent manner. This system enables, for the first time, reliable pregnancy stage-specific induction of gene expression in the placenta and live monitoring of gene expression during pregnancy. It will be widely applicable to studies of both placental development and pregnancy, and the site-specific Tet-On G3 mouse will be valuable for studies in a broad range of tissues.

Differentiation Potential of Human Induced Pluripotent Stem Cells (iPSCs) to Nucleus Pulposus-Like Cells in Vitro

Introduction Autologous or allogeneic cell delivery to the herniated or degenerated intervertebral disk may promote tissue regeneration and arrest degeneration1–4. Induced pluripotent stem cells (iPSCs) derived from the patient's somatic cells represent an attractive cell source, having the potential to differentiate into various cell types5. To date, no studies have demonstrated that human iPSCs can differentiate into nucleus pulposus (NP) cells. We have previously shown that both human umbilical cord mesenchymal stromal cells and mouse iPSCs were able to display many NP-like cell markers after cultured in a laminin-rich 3D culture system for inducing differentiation6,7. The goal of this study is to evaluate whether human iPSCs cultured in this similar laminin-rich environment can also express a unique NP-like cell phenotype. Materials and Methods Cell Generation and Characterization iPSCs were generated from human embryonic dermal fibroblasts through transient inducible over-expression of transcription factors (OCT4, SOX2, KLF4 and MYC) by a lentiviral-based gene delivery system (a poly-cystronic vector with the Reverse Tetracycline Transactivator (M2rtTA) and doxycycline). For the derived colonies, immuno-cytochemistry was performed using a pluripotent markers kit (OCT4, SOX2, SSEA-4, TRA1-60, TRA1-81 and alkaline phosphatase, Applied StemCell). Selected iPSC colonies were maintained in culture upon a PMEF feeder layer. Cell Differentiation Undifferentiated iPSCs were seeded (106/well, Transwell) on wells precoated with Matrigel to generate a soft laminin-rich matrix6,7. Cells were cultured in conditioned medium collected from porcine NP tissues (DMEM + 1% ITS)8 for up to 28 days. Flow Cytometry Analysis Undifferentiated iPSCs were incubated with the antibodies against CD24, CD31, CD90, integrin subunits a3 (CD49c), a6 (CD49f), and b1 (CD29). For intracellular expression of transcription factors (Brachyury, OCT4), cells were permeabilized before incubation with primary antibodies. Cells were analyzed for fluorescence (Accuri C6) to quantify the percent of positively labeled cells. Immunohistochemistry Differentiated cells were harvested for cryo-sectioning. Cell morphology and proteoglycan synthesis was assessed by histological staining (H&amp;E and Safranin O). Expression of NP markers was evaluated by immunostaining for matrix proteins (type II collagen (COL II), laminin 10 (LM511)), laminin-related receptors (CD239, integrin subunits a3, a6, b1, b4), and other markers (vimentin (VIM), cytokeratin 8 (KRT8), N-cadherin, CD24), as well as non-NP-markers (type I collagen, E-cadherin). Results iPSCs proliferated and formed colonies on PMEF feeder layers similar to embryonic stem cells ( Fig. 1A ). During proliferation, iPSC colonies expressed many typical human pluripotent markers (i.e. SOX2, TRA1-60 in Fig. 1A ), and expressed integrin (α3, α6 and β1 subunit) proteins, other NP markers (CD24 and Brachyury), as well as MSC markers (CD29, CD90). Once cultured under soft laminin-rich matrix conditions, iPSCs were shown to adopt a cell clustering morphology. At day 28 of differentiation, iPSCs expressed NP-related matrix proteins (laminin 10, LM511; type II collagen and proteoglycans as indicated by Saf O, Fig. 1B ), LM511 specific receptors (CD239, integrin subunit a3, a6 and b4, Fig. 1 B), as well as many NP markers (CD24, vimentin, cytokeratin 8 and N-cadherin, Fig. 1B ); it is noteworthy that type I collagen and E-cadherin were not expressed. [Figure: see text] Conclusion Our study evaluated a novel cell source that has potential use for cellular therapy in the IVD. Results demonstrate that native NP environment factors (soft laminin-rich matrix and secreted soluble factors) are able to promote human iPSC differentiation into a cell type exhibiting many NP-like markers and morphology. Future studies will focus on selection of NP-genic progenitor cells from these hiPSCs for IVD cellular therapy. Acknowledgments Supported by NIH R01AR057410, R01EB002263, R01AR047442,AOSpine Foundation and FAMRI Young Clinical Scientist award. I confirm having declared any potential conflict of interest for all authors listed on this abstract No Disclosure of Interest None declared Nishimura, et al. Spine 1998;23:1531 Okuma, et al. Journal of Orthopaedic Research 2000;18:988 Meisel, et al. European Spine Journal 2006;15(S3):397 Sakai. Orthopaedic Clinics of North America. 2011;42:555 Yamanaka. Cell 2009;137:13 Chon, et al. Trans ORS 2011;36:600 Jing, et al. Tans ORS 2012;37: 31 Purmessur, et al. Arthritis Research Therapy 2011;13:R81

Ocular gene delivery using lentiviral vectors

Substantial advances in our understanding of lentivirus lifecycles and their various constituent proteins have permitted the bioengineering of lentiviral vectors now considered safe enough for clinical trials for both lethal and non-lethal diseases. They possess distinct properties that make them particularly suitable for gene delivery in ophthalmic diseases, including high expression, consistent targeting of various post-mitotic ocular cells in vivo and a paucity of associated intraocular inflammation, all contributing to their ability to mediate efficient and stable intraocular gene transfer. In this review, the intraocular tropisms and therapeutic applications of both primate and non-primate lentiviral vectors, and how the unique features of the eye influence these, are discussed. The feasibility of therapeutic targeting using these vectors in animal models of both anterior and posterior ophthalmic disorders has been established, and has, in combination with substantial progress in enhancing lentiviral vector bio-safety over the past two decades, paved the way for the first human ophthalmic clinical trials using lentivirus-based gene transfer vectors.

Noninvasive and real‐time monitoring of the therapeutic response of tumors in vivo with an optimized hTERT promoter

Telomerase is commonly recognized as an effective anticancer target. The human telomerase reverse transcriptase (hTERT), the rate-limiting component of telomerase, is expressed in most malignant tumors, but it is not found in most normal somatic cells. Here, we report a real-time and noninvasive method to monitor tumor response to a lentivirus-based hTERT-conditional suicidal gene therapy. In this study, we constructed a lentivirus system in which an optimized hTERT promoter was used to drive the expression of the cytosine deaminase (CD) gene, one of the suicide genes, and a green fluorescent protein (GFP) reporter gene (pLenti-CD/GFP). The lentivirus was used to infect telomerase-positive or telomerase-negative cell lines. In vitro and in vivo experiments were conducted to analyze the dynamic processes of exogenous gene expression noninvasively in cell culture and living animals in real time via optical imaging. The lentivirus was able to express the CD gene and GFP in telomerase-positive tumor cells and significantly decrease cell proliferation after the use of prodrug 5-flucytosine. However, it could not express GFP and CD in telomerase-negative cell lines, nor could it induce any suicidal effect in those cells. The in vivo study showed that telomerase-positive tumors can be visualized after intratumor injection of the lentivirus in tumor-bearing nude mice via an optical imaging system. Significant tumor growth suppression was observed in telomerase-positive tumors. Collectively, this technology provides a valuable, noninvasive method to evaluate the real-time therapeutic response of tumors in vivo.

Genetically encoding unnatural amino acids in neural stem cells and optically reporting voltage-sensitive domain changes in differentiated neurons.

Although unnatural amino acids (Uaas) have been genetically encoded in bacterial, fungal, and mammalian cells using orthogonal transfer RNA (tRNA)/aminoacyl-tRNA synthetase pairs, applications of this method to a wider range of specialized cell types, such as stem cells, still face challenges. While relatively straightforward in stem cells, transient expression lacks sufficient temporal resolution to afford reasonable levels of Uaa incorporation and to allow for the study of the longer term differentiation process of stem cells. Moreover, Uaa incorporation may perturb differentiation. Here, we describe a lentiviral-based gene delivery method to stably incorporate Uaas into proteins expressed in neural stem cells, specifically HCN-A94 cells. The transduced cells differentiated into neural progenies in the same manner as the wild-type cells. By genetically incorporating a fluorescent Uaa into a voltage-dependent membrane lipid phosphatase, we show that this Uaa optically reports the conformational change of the voltage-sensitive domain in response to membrane depolarization. The method described here should be generally applicable to other stem cells and membrane proteins.