Adenovirus Gene Transfer Vectors Research Articles

S100A11 Promotes Liver Steatosis via FOXO1-Mediated Autophagy and Lipogenesis.

Background & AimsNonalcoholic fatty liver disease (NAFLD) is becoming a severe liver disorder worldwide. Autophagy plays a critical role in liver steatosis. However, the role of autophagy in NAFLD remains exclusive and under debate. In this study, we investigated the role of S100 calcium binding protein A11 (S100A11) in the pathogenesis of hepatic steatosis.MethodsWe performed liver proteomics in a well-established tree shrew model of NAFLD. The expression of S100A11 in different models of NAFLD was detected by Western blot and/or quantitative polymerase chain reaction. Liver S100A11 overexpression mice were generated by injecting a recombinant adenovirus gene transfer vector through the tail vein and then induced by a high-fat and high-cholesterol diet. Cell lines with S100a11 stable overexpression were established with a recombinant lentiviral vector. The lipid content was measured with either Bodipy staining, Oil Red O staining, gas chromatography, or a triglyceride kit. The autophagy and lipogenesis were detected in vitro and in vivo by Western blot and quantitative polymerase chain reaction. The functions of Sirtuin 1, histone deacetylase 6 (HDAC6), and FOXO1 were inhibited by specific inhibitors. The interactions between related proteins were analyzed by a co-immunoprecipitation assay and immunofluorescence analysis.ResultsThe expression of S100A11 was up-regulated significantly in a time-dependent manner in the tree shrew model of NAFLD. S100A11 expression was induced consistently in oleic acid–treated liver cells as well as the livers of mice fed a high-fat diet and NAFLD patients. Both in vitro and in vivo overexpression of S100A11 could induce hepatic lipid accumulation. Mechanistically, overexpression of S100A11 activated an autophagy and lipogenesis process through up-regulation and acetylation of the transcriptional factor FOXO1, consequently promoting lipogenesis and lipid accumulation in vitro and in vivo. Inhibition of HDAC6, a deacetylase of FOXO1, showed similar phenotypes to S100A11 overexpression in Hepa 1–6 cells. S100A11 interacted with HDAC6 to inhibit its activity, leading to the release and activation of FOXO1. Under S100A11 overexpression, the inhibition of FOXO1 and autophagy could alleviate the activated autophagy as well as up-regulated lipogenic genes. Both FOXO1 and autophagy inhibition and Dgat2 deletion could reduce liver cell lipid accumulation significantly.ConclusionsA high-fat diet promotes liver S100A11 expression, which may interact with HDAC6 to block its binding to FOXO1, releasing or increasing the acetylation of FOXO1, thus activating autophagy and lipogenesis, and accelerating lipid accumulation and liver steatosis. These findings indicate a completely novel S100A11-HDAC6-FOXO1 axis in the regulation of autophagy and liver steatosis, providing potential possibilities for the treatment of NAFLD.

Prevention of Corneal Neovascularization by Adenovirus Encoding Human Vascular Endothelial Growth Factor Soluble Receptor (s-VEGFR1) in Lacrimal Gland.

The aims of this study were (1) to determine the efficacy of adenovirus vector serotype 5 (Ad) encoding human soluble VEGF receptor 1 (s-VEGFR1) gene transfer to the lacrimal gland (LG); (2) to investigate whether expression of s-VEGFR1 prevents corneal neovascularization (CNV) induced by alkali burns; and (3) to evaluate the safety of the procedure. AdVEGFR1 vectors (25 μL, 1 × 1010 pfu/mL) were injected in the right LGs of rats and were compared with AdNull vector (25 μL, 1 × 1010 pfu/mL) or 25 μL of saline (Control) before cornea alkali burns with 1 M NaOH. After 7 days, CNV was documented at the slit lamp. Tear secretion was measured with phenol red threads. The animals were tested for s-VEGFR1 mRNA and protein in the LG by quantitative (q)PCR and immunohistochemistry staining, respectively. qPCR was used to compare the mRNA levels of IL-1β, IL-6, and TNF-α in the LG and ipsilateral trigeminal ganglion (TG). Ad-VEGFR1 transfected 83% (10/12) of the rats. VEGFR1 was present in LG acinar cells. CNV was prevented in 9 of 12 animals in the Ad-VEGFR1 group, compared with the Ad-Null (3:10) and Control groups (1:10) (P = 0.0317). The tear secretion and cytokine mRNA levels in the LG and TG were similar in all three groups (P > 0.05). Adenoviral vector gene transfer was safe for LG structure and function. The LG as the target tissue showed local expression of human s-VEGFR1, and CNV was prevented in most of the eyes exposed to alkali burns.

Combined Genetic and Chemical Capsid Modifications of Adenovirus-Based Gene Transfer Vectors for Shielding and Targeting.

Adenovirus vectors are potent tools for genetic vaccination and oncolytic virotherapy. However, they are prone to multiple undesired vector-host interactions, especially after in vivo delivery. It is a consensus that the limitations imposed by undesired vector-host interactions can only be overcome if defined modifications of the vector surface are performed. These modifications include shielding of the particles from unwanted interactions and targeting by the introduction of new ligands. The goal of the protocol presented here is to enable the reader to generate shielded and, if desired, retargeted human adenovirus gene transfer vectors or oncolytic viruses. The protocol will enable researchers to modify the surface of adenovirus vector capsids by specific chemical attachment of synthetic polymers, carbohydrates, lipids, or other biological or chemical moieties. It describes the cutting-edge technology of combined genetic and chemical capsid modifications, which have been shown to facilitate the understanding and overcoming of barriers for in vivo delivery of adenovirus vectors. A detailed and commented description of the crucial steps for performing specific chemical reactions with biologically active viruses or virus-derived vectors is provided. The technology described in the protocol is based on the genetic introduction of (naturally absent) cysteine residues into solvent-exposed loops of adenovirus-derived vectors. These cysteine residues provide a specific chemical reactivity that can, after production of the vectors to high titers, be exploited for highly specific and efficient covalent chemical coupling of molecules from a wide variety of substance classes to the vector particles. Importantly, this protocol can easily be adapted to perform a broad variety of different (non-thiol-based) chemical modifications of adenovirus vector capsids. Finally, it is likely that non-enveloped virus-based gene transfer vectors other than adenovirus can be modified from the basis of this protocol.

Intravesical rad-IFNα/Syn3 for patients with high-grade, bacillus Calmette-Guérin (BCG) refractory or relapsed non-muscle invasive bladder cancer: A phase II randomized study.

279 Background: While bacillus Calmette-Guérin (BCG) is the most effective intravesical treatment for reducing recurrence and progression for high-risk non-muscle invasive bladder cancer (NMIBC), many patients are either refractory to treatment or relapse. We assessed the efficacy and safety of rAd-IFNα/Syn 3 (Instiladrin, FKD Finland), a replication deficient recombinant adenovirus gene transfer vector, for patients with high-grade (HG) BCG refractory or relapsed NMIBC. Methods: In this open-label, US multicenter (n=13), parallel-arm Phase II study (NCT01687244), 43 patients with HG BCG refractory or relapsed NMIBC were randomized 1:1 to receive intravesical rAd-IFNα/Syn3 at 1x1011 or 3x1011viral particles/mL. Patients responding at months 3, 6, and 9 were re-treated at months 4, 7, and 10. Most patients (n=21) had a primary tumor classification of carcinoma in situ (CIS); 9 had both CIS and Ta/T1 disease and 10 had Ta/T1 disease alone. The primary endpoint was 12-month HG recurrence-free survival (RFS). All patients receiving at least one dose were included in efficacy and safety analyses. Results: Forty patients received rAd-IFNα/Syn3 (1 x 1011 vp/mL: n=21; 3 x 1011vp/mL: n=19) between November 2012 and April 2015. Fourteen patients (35.0%; 90% CI 22.6%, 49.2%) remained free of HG tumor recurrence 12 months after initial treatment. Comparable 12 month HG RFS was noted between dosage arms, as well as between patients with refractory and relapsed NMIBC. Interestingly, the 12 month HG recurrence-free survival for patients with Ta/T1-only disease was 50% (5/10). rAd-IFNα/Syn3 was well-tolerated, with no Grade 5 adverse events (AEs), one Grade 4 AE (COPD; unrelated to treatment), and no patient discontinuing treatment due to an adverse event. The most frequently reported AEs were micturition urgency (n=16), dysuria and pollakiuria (n=13 each), fatigue (n=9), and nocturia (n=10). Conclusions: rAd-IFNα/Syn3 was well tolerated and demonstrated promising efficacy for patients with HG NMIBC after BCG therapy who are unable or unwilling to undergo radical cystectomy. A phase III trial utilizing this novel agent is ongoing. Clinical trial information: 01687244.

Enhancing Adenoviral-Mediated Gene Transfer and Expression to Endometrial Cells.

Our aim was to screen a panel of modified adenoviral gene transfer vectors to identify those which can sustain high gene expression in human endometrial cells. Normal endometrial stromal cell cultures were established from endometrial lining of hysterectomy specimens performed for benign gynecologic indications. Human endometrial stromal cells were transfected by modified adenoviruses expressing luciferase reporter gene. Luciferase activity mediated by each virus was expressed as a percentage of adenovirus serotype 5 (Ad5-CMV-luc) activity. The 2-tailed Student t test was used to compare data. At a multiplicity of infection (MOI) of 10 pfu/cell, of the transductionally modified adenoviruses, adenovirus-RGD (Ad-RGD-luc) mediated highest level of endometrial cell transduction with transgene expression around 4 times higher when compared to Ad5 (P < .001). Of the transcriptionally targeted adenoviruses, adenovirus under secretory leukocyte protease inhibitor promoter (Ad-SLPI-luc) and adenovirus under heparanase promoter (Ad-heparanase-luc)-mediated luciferase activation were 5.8- and 4.3-folds higher than Ad5-CMV-luc, respectively (P = .02 and .03, respectively). At MOI of 50 pfu/cell, Ad-RGD-luc and AD-SLPI-luc mediated significantly higher gene transfer efficiency compared to Ad5-CMV-luc (P values < .001, for each virus). Ad-heparanase-luc achieved higher gene activity, but difference was not significant (P = .1). Ad-SLPI-luc, at low viral dose (10 pfu/ cell), mediated gene expression effect comparable to Ad5-CMV-luc at a high dose (50 pfu/cell), with no significant difference. We conclude that when compared to the wild-type adenovirus, Ad-RGD-luc, Ad-SLPI-luc, and Ad-heparanase-luc mediate higher reporter gene activity in endometrial cells and can work as effective gene transfer vectors in gene therapy applications to the endometrium.

36. Translation of an Adenovirus-Based Cocaine Vaccine dAd5GNE to a Clinical Trial

dAd5GNE, an anti-cocaine vaccine based on a disrupted serotype 5 adenovirus gene transfer vector covalently conjugated to the cocaine analog GNE, evokes high titers of high affinity anti-cocaine antibodies that prevent cocaine from reaching its cognate receptor in the CNS. dAd5GNE has been shown to be effective in preclinical efficacy studies in mice, rats and nonhuman primates. In order to translate dAd5GNE to the clinic, this study focused on optimizing the choice of adjuvant, timing of vaccination regimen and dose. Adjuvant. To evaluate vaccine formulation, the adjuvants Adjuplex and Alum were assessed at a fixed dose to identify the formulation that evoked the fastest and highest titer response. BALB/c mice vaccinated with dAd5GNE/Adjuplex demonstrated high serum anti-cocaine antibody titers (>5×105) after a single administration while animals vaccinated with dAd5GNE/Alum required multiple injections to achieve high titers, and mice vaccinated with dAd5GNE/PBS plateaued at a lower titer. Therefore, a formulation based on the Adjuplex adjuvant was chosen for the clinical protocol. Timing. The measurement of titer half-life in nonhuman primates following individual vaccinations at variable intervals was used to inform the timing between vaccine boosts. The anti-cocaine antibody titer half-life in 8 dAd5GNE/Adjuplex vaccinated nonhuman primates was assayed regularly over the course of 1 yr and the titer half-life was calculated following each administration. The average half-life for all animals was 4.0 ± 0.2 wk. Continued cocaine use did not impact the titer half-life. These results indicate that a 4 wk interval for vaccinations is necessary to maintain the high titer anti-cocaine antibody levels. Dose. We measured the dose response in mice to provide the baseline for a phase I/II clinical trial and identified the range of doses that bracket the anticipated minimum effective dose and maximum tolerated dose. Doses from 0.04-40 μg of the vaccine were evaluated in BALB/c for the capacity to evoke antibody titers and to minimize access of radiolabelled cocaine to the CNS. Doses at and above 4 µg produced high anti-cocaine antibody titers above the threshold for efficacy (>5×105) which substantially reduced cocaine levels in the brain (p<0.0001 vs PBS). Using a modified weight adjusted dose from these results, the vaccine doses to be evaluated in the clinical trial were determined to be 100 to 1000 µg. The results of these studies combine to provide the specifications for critical vaccine design parameters required to translate the anti-cocaine vaccine to evaluation in a clinical trial. Based on these specifications the FDA has allowed the Adjuplex-formulated dAd5GNE vaccine to proceed to clinical study with a monthly vaccination regimen in recovering cocaine addicts.

Abstract 221: MitoROMK Expression Mitigates the Cellular Response to Oxidative Stress

We have recently reported that mitoROMK overexpression protects against, while knockdown of native ROMK exacerbates, oxidative stress-induced cell death and have proposed mitoROMK as the molecular correlate of the cardioprotective mitoKATP channel (Foster, Ho, et al Circ Res 2012). In order to better understand the mechanisms of protection, here we examined how alterations of ROMK expression affect glutathione redox responses during acute oxidative stress, utilizing cytoplasmic or mitochondrially targeted, genetically-encoded GSSG sensors. The tandem protein construct Grx1-roGFP2, a fusion of human glutaredoxin and redox sensitive GFP (Gutscher et al, Nat Methods 2008) was incorporated into adenoviral gene transfer vectors for expression in cardiac-derived H9C2 cells. Cells were subjected to treatment with the Complex III inhibitor Antimycin A (50µM) to induce endogenous mitochondrial reactive oxygen species (ROS) production, and changes in the glutathione redox potential were determined ratiometrically. MitoROMK overexpressing cells showed a significantly lower rate of mitochondrial matrix GSH oxidation secondary to H2O2 scavenging compared to both control and ROMK knockdown cell lines, indicating enhanced ability to maintain ROS balance. Increases in cytoplasmic GSSG were also blunted by mitoROMK overexpression. To determine if ROMK expression affected ROS-induced mitochondrial calcium overload, we also employed the mitochondrially-targeted ratiometric Ca2+ probe GEM-GECO (Zhao et al Science 2011). The same Antimycin A challenge led to large mitochondrial Ca2+ increase in control and knockdown cell lines, but this response was significantly suppressed in mitoROMK overexpressing cells. This could not be explained by differences in baseline mitochondrial membrane potential (ΔΨm; determined using the potentiometric probe tetramethylrhodamine methyl ester), because both control cells and ROMK overexpressors showed similar ΔΨm, while ROMK knockdown cells were more depolarized. The results suggest that enhanced cell survival in mitoROMK overexpressing cells is mediated by improved mitochondrial ROS balance and calcium handling, providing a key molecular link between mitochondrial K+ channels and cardioprotection.

Novel Cocaine Vaccine Linked to a Disrupted Adenovirus Gene Transfer Vector Blocks Cocaine Psychostimulant and Reinforcing Effects

Immunotherapy is a promising treatment for drug addiction. However, insufficient immune responses to vaccines in most subjects pose a challenge. In this study, we tested the efficacy of a new cocaine vaccine (dAd5GNE) in antagonizing cocaine addiction-related behaviors in rats. This vaccine used a disrupted serotype 5 adenovirus (Ad) gene transfer vector coupled to a third-generation cocaine hapten, termed GNE (6-(2R,3S)-3-(benzoyloxy)-8-methyl-8-azabicyclo [3.2.1] octane-2-carboxamido-hexanoic acid). Three groups of rats were immunized with dAd5GNE. One group was injected with (3)H-cocaine, and radioactivity in the blood and brain was determined. A second group was tested for cocaine-induced locomotor sensitization. A third group was examined for cocaine self-administration, extinction, and reinstatement of responding for cocaine. Antibody titers were determined at various time-points. In each experiment, we added a control group that was immunized with dAd5 without a hapten. The vaccination with dAd5GNE produced long-lasting high titers (>10(5)) of anti-cocaine antibodies in all of the rats. The vaccination inhibited cocaine-induced hyperlocomotor activity and sensitization. Vaccinated rats acquired cocaine self-administration, but they showed less motivation to self-administer cocaine under a progressive-ratio schedule than control rats. When cocaine was not available in a session, control rats exhibited 'extinction burst' responding, whereas vaccinated rats did not. Moreover, when primed with cocaine, vaccinated rats did not reinstate responding, suggesting a blockade of cocaine-seeking behavior. These data strongly suggest that our dAd5GNE vector-based vaccine may be effective in treating cocaine abuse and addiction.

Rescue, Amplification, and Large-Scale Production of Helper-Dependent Adenoviral Vectors

INTRODUCTIONThis protocol describes in detail the rescue, amplification, and large-scale production of helper-dependent adenoviral vectors (HDAds) for gene transfer and gene therapy.Production of HDAd can be divided into three parts: (1) rescue, which involves converting pHDAd (plasmid form) to HDAd (viral form); (2) amplification, in which the amount of HDAd is increased by serial coinfections (passages) of the producer cells with the HDAd and the helper virus; and (3) large-scale production to generate large quantities of HDAd. HDAds (also referred to as gutless, gutted, mini, fully deleted, high-capacity, Δ, pseudo) are deleted of all virus-coding sequences. HDAds retain the advantages of early-generation Ad vectors including high-efficiency in vivo transduction and high-level transgene expression. However, the absence of viral gene expression in transduced cells permits long-term transgene expression in the absence of chronic toxicity. Moreover, the deletion of the viral sequences permits a cloning capacity of ∼37 kb. This allows for the delivery of whole-genomic loci, multiple transgenes, and large cis-acting elements to enhance, prolong, and regulate transgene expression. In addition, because the vector genome exists episomally in transduced cells, the risks of germ-line transmission and insertional mutagenesis leading to oncogenic transformation are negligible.

Cocaine Analog Coupled to Disrupted Adenovirus: A Vaccine Strategy to Evoke High-titer Immunity Against Addictive Drugs

Based on the concept that anticocaine antibodies could prevent inhaled cocaine from reaching its target receptors in the brain, an effective anticocaine vaccine could help reverse cocaine addiction. Leveraging the knowledge that E1(-)E3(-) adenovirus (Ad) gene transfer vectors are potent immunogens, we have developed a novel vaccine platform for addictive drugs by covalently linking a cocaine analog to the capsid proteins of noninfectious, disrupted Ad vector. The Ad-based anticocaine vaccine evokes high-titer anticocaine antibodies in mice sufficient to completely reverse, on a persistent basis, the hyperlocomotor activity induced by intravenous administration of cocaine.

Respiratory mucosal immunization with adenovirus gene transfer vector induces helper CD4 T cell‐independent protective immunity

Virus-vectored vaccine is a powerful activator of CD8 T cell-mediated immunity and is especially amenable to respiratory mucosal immunization, offering hopes for use in humans with diminished helper CD4 T cell function. However, whether virus-mediated mucosal immunization can produce immune protective CD8 T cells without the CD4 T cell help remains to be investigated. We used a replication-deficient adenovirus vector expressing an Mycobacterium tuberculosis antigen Ag85A for intranasal vaccination and evaluated its effect on CD8 T cell activation and protection in mice depleted of CD4 T cells. Intranasal vaccination of CD4 T cell-depleted mice led to suboptimal generation of Ag-specific tetramer(+) or interferon (IFN)-gamma-producing CD8 T cells in the lung and spleen but this was observed mainly at the early time after vaccination. Reduced CD8 T cell priming was also accompanied by decreased CD8 T cell responses (CTL). Nevertheless, the ratio of Ag-specific CD8 T cells to IFN-gamma-producing CD8 T cells in CD4 T cell-depleted hosts remained comparable to that in CD4 T cell-competent hosts. Furthermore, the 'unhelped' CD8 T cells also displayed a similar immune phenotype as the 'helped' counterparts. The animals with 'unhelped' CD8 T cells were as well-protected from pulmonary M. tuberculosis challenge as those with 'helped' CD8 T cells in the absence of CD4 T cells. The data obtained in the present study suggest that the fully immune protective CD8 T cells can still be generated by respiratory mucosal viral-mediated immunization without CD4 T cells and that CD8 T cells, 'helped' or 'unhelped', can confer significant protection against pulmonary tuberculosis independent of CD4 T cells.

Protective Immunity Against a Lethal RespiratoryYersinia pestisChallenge Induced by V Antigen or the F1 Capsular Antigen Incorporated into Adenovirus Capsid

The aerosol form of the bacterium Yersinia pestis causes pneumonic plague, a rapidly fatal disease that is a biothreat if deliberately released. At present, no plague vaccines are available for use in the United States, but subunit vaccines based on the Y. pestis V antigen and F1 capsular protein show promise when administered with adjuvants. In the context that adenovirus (Ad) gene transfer vectors have a strong adjuvant potential related to the ability to directly infect dendritic cells, we hypothesized that modification of the Ad5 capsid to display either the Y. pestis V antigen or the F1 capsular antigen on the virion surface would elicit high V antigen- or F1-specific antibody titers, permit boosting with the same Ad serotype, and provide better protection against a lethal Y. pestis challenge than immunization with equivalent amounts of V or F1 recombinant protein plus conventional adjuvant. We constructed AdYFP-pIX/V and AdLacZ-pIX/F1, E1(-), E3(-) serotype 5 Ad gene transfer vectors containing a fusion of the sequence for either the Y. pestis V antigen or the F1 capsular antigen to the carboxy-terminal sequence of pIX, a capsid protein that can accommodate the entire V antigen (37 kDa) or F1 protein (15 kDa) without disturbing Ad function. Immunization with AdYFP-pIX/V followed by a single repeat administration of the same vector at the same dose resulted in significantly better protection of immunized animals compared with immunization with a molar equivalent amount of purified recombinant V antigen plus Alhydrogel adjuvant. Similarly, immunization with AdLacZ-pIX/F1 in a prime-boost regimen resulted in significantly enhanced protection of immunized animals compared with immunization with a molar-equivalent amount of purified recombinant F1 protein plus adjuvant. These observations demonstrate that Ad vaccine vectors containing pathogen-specific antigens fused to the pIX capsid protein have strong adjuvant properties and stimulate more robust protective immune responses than equivalent recombinant protein-based subunit vaccines administered with conventional adjuvant, suggesting that F1-and/or V-modified capsid Ad-based recombinant vaccines should be considered for development as anti-plague vaccines.

Genomic integration of adenoviral gene transfer vectors following transduction of fertilized mouse oocytes

Adenoviral vectors (AdV) are popular tools to deliver foreign genes into a wide range of cells. They have also been used in clinical gene therapy trials. Studies on AdV-mediated gene transfer to mammalian oocytes and transmission through the germ line have been reported controversially. In the present study we investigated whether AdV sequences integrate into the mouse genome by microinjecting AdV into the perivitelline space of fertilized oocytes. We applied a newly developed PCR technique (HiLo-PCR) for identification of chromosomal junctions next to the integrated AdV. We demonstrate that mouse oocytes can be transduced by different recombinant adenoviral vectors (first generation and gutless). In one transgenic mouse line using the first generation adenoviral vector, the genome has integrated into a highly repetitive cluster located on the Y chromosome. While the transgene (GFP) was expressed in early embryos, no expression was detected in adult transgenic mice. The use of gutless AdV resulted in expression of the transgene, albeit the vector was not transmitted to progeny. These results indicate that under optimized conditions fertilized mouse oocytes are transduced by AdV and give rise to transgenic founder animals. Therefore, adequate precautions should be taken in gene therapy protocols of reproductive patients since transduction of oocytes or early embryos and subsequent chromosomal integration cannot be ruled out entirely.

Assessment of CAR- or CD46-Dependent Adenoviral Vector-Mediated TRAIL Gene Therapy in Clinical Adenocarcinoma Lung Cancer Cells

Adenoviral vector-mediated transfer of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can be a powerful approach to lung cancer therapy. However, the efficiency of adenoviral vector gene transfer and the sensitivity to TRAIL-induced apoptosis in the context of adenoviral vector gene transfer have yet to be characterized in primary lung cancers. In this study, we investigated the expression of adenoviral receptor CD46 expression in primary lung cancer cells. In contrast to previous reports on enhanced CD46 expression in various types of cancer cells, we show a significantly higher CD46 expression in lung adenocarcinomas compared to lung squamous cell carcinomas. Using Ad5-GFP and Ad5F35-GFP vectors, we demonstrated an improved gene transfer efficiency in primary lung cancer cells by the Ad5F35 vector. The apoptosis induction effect mediated by Ad5-TRAIL and Ad5F35-TRAIL vector gene transfer was compared in cells from 10 lung adenocarcinomas. Of 5 lung cancers in which apoptosis was induced, 2 had an enhanced effect by Ad5F35-TRAIL vector gene transfer compared to Ad5-GFP. Thus, these results indicate a method to identify TRAIL-sensitive primary lung cancers, which will also facilitate the analysis of resistance mechanisms in lung cancers.

New ectopic vestibular hair cell-like cells induced by Math1 gene transfer in postnatal rats

Overexpression of Math1 has been demonstrated to induce robust ectopic new hair cells in greater epithelial ridge (GER) and lesser epithelial ridge (LER) of the postnatal rats' cochlear in vitro. In spite of the similarities between cochlear and vestibular epithelia in origin and structure, no similar results have been reported in the non-sensory region of vestibular epithelia in vitro. In the study, the adenoviral vectors inserted with Math1 gene were constructed to examine their effect on vestibular epithelia in postnatal rats. In vivo, the adenovirus vectors administered in vestibular perilymphatic or endolymphatic space, their transduction efficiency and other indexes are different. We set up a culture construction to simulate and show the differences. In the study, we also developed a new dissection protocol to be quick to harvest the whole maculae and cristae. The new ectopic vestibular hair cell-like cells induced by overexpression of Math1, were found to appear in the non-sensory region of the postnatal rats' vestibular epithelia as observed in the cochlear, and the number of the new cells was different when a different virus administration was simulated in vestibular perilymphatic or endolymphatic space, suggesting that the cells found could have the capability to differentiate into new hair cells. Our study might pave the way for further in vivo studies on vestibular Math1 adenoviral vector gene transfer.

962. Enhanced Protection Against a Lethal Yersinia pestis Challenge by an Affinity Matured Anti-V Antigen Monoclonal Antibody Delivered with an Adenovirus Gene Transfer Vector

962. Enhanced Protection Against a Lethal Yersinia pestis Challenge by an Affinity Matured Anti-V Antigen Monoclonal Antibody Delivered with an Adenovirus Gene Transfer Vector

Emphysema Mediated by Lung Overexpression of ADAM10

Cigarette smoking is the major risk factor for emphysema, a disorder of the lung parenchyma characterized by destruction of the alveolar walls. Current concepts of the pathogenesis of emphysema hold that the destruction of the lung parenchyma results, in part, from a local imbalance of proteases and antiproteases. Based on the knowledge that human alveolar macrophages express ADAM 10, a protease capable of destroying basement membrane collagen but not previously implicated in emphysema, we used adenovirus-mediated lung expression of ADAM 10 in a mouse model to assess whether an increased burden of ADAM 10 was capable of inducing emphysema. To assess this, the human ADAM 10 cDNA under control of a constitutive promoter was inserted into an adenovirus gene transfer vector (AdhADAMlO), and the vector (10(11) particle units) administered to the respiratory tract of wild type C57BI/6 mice. Lung levels of ADAM 10 mRNA and protein were upregulated following AdhADAMlO administration. After 8 weeks, quantitative morphometry of the lung parenchyma demonstrated that AdhADAMlO administration induced emphysema (mean linear intercept of 60.6 + 1.3 microm compared with 55.6 + 0.8 in mice treated with a control vector, p < 0.003). These results suggest a role of ADAM 10 in the pathogenesis of emphysema, adding to the list of proteases expressed in the lung that are capable of contributing to the development of lung destruction.

Adenovirus-Mediated Delivery of an Anti-V Antigen Monoclonal Antibody Protects Mice against a Lethal Yersinia pestis Challenge

Pneumonic plague, caused by inhalation of Yersinia pestis, represents a major bioterrorism threat for which no vaccine is available. Based on the knowledge that genetic delivery of monoclonal antibodies (MAbs) with adenovirus (Ad) gene transfer vectors results in rapid, high-level antibody expression, we evaluated the hypothesis that Ad-mediated delivery of a neutralizing antibody directed against the Y. pestis V antigen would protect mice against a Y. pestis challenge. MAbs specific for the Y. pestis V antigen were generated, and the most effective in protecting mice against a lethal intranasal Y. pestis challenge was chosen for further study. The coding sequences for the heavy and light chains were isolated from the corresponding hybridoma and inserted into a replication-defective serotype 5 human Ad gene transfer vector (AdalphaV). Western analysis of AdalphaV-infected cell supernatants demonstrated completely assembled antibodies reactive with V antigen. Following AdalphaV administration to mice, high levels of anti-V antigen antibody titers were detectable as early as 1 day postadministration, peaked by day 3, and remained detectable through a 12-week time course. When animals that received AdalphaV were challenged with Y. pestis at day 4 post-AdalphaV administration, 80% of the animals were protected, while 0% of control animals survived (P < 0.01). Ad-mediated delivery of a V antigen-neutralizing antibody is an effective therapy against plague in experimental animals and could be developed as a rapidly acting antiplague therapeutic.

Identification of an exonic splicing silencer in exon 6A of the human VEGF gene

BackgroundThe different isoforms of vascular endothelial growth factor (VEGF) play diverse roles in vascular growth, structure and function. Alternative splicing of the VEGF gene results in the expression of three abundant isoforms: VEGF121, VEGF165 and VEGF189. The mRNA for VEGF189 contains the alternatively spliced exon 6A whereas the mRNA for VEGF165 lacks this exon. The objective of this study was to identify the cis elements that control utilization of exon 6A. A reporter minigene was constructed (pGFP-E6A) containing the coding sequence for GFP whose translation was dependent on faithful splicing for removal of the VEGF exon 6A. To identify cis-acting splicing elements, sequential deletions were made across exon 6A in the pGFP-E6A plasmid.ResultsA candidate cis-acting exonic splicing silencer (ESS) comprising nucleotides 22-30 of exon 6A sequence was identified corresponding to the a silencer consensus sequence of AAGGGG. The function of this sequence as an ESS was confirmed in vivo both in the context of the reporter minigene as a plasmid and in the context of a longer minigene with VEGF exon 6A in its native context in an adenoviral gene transfer vector. Further mutagenesis studies resulted in the identification of the second G residue of the putative ESS as the most critical for function.ConclusionThis work establishes the identity of cis sequences that regulate alternative VEGF splicing and dictate the relative expression levels of VEGF isoforms.

Over-expression of the potassium channel Kir2.3 using the dopamine-1 receptor promoter selectively inhibits striatal neurons

Dysfunction of basal ganglia circuits underlies a variety of movement disorders and neuropsychiatric conditions. Selective control of the electrical activity of striatal outflow pathways by manipulation of ion channel function presents a novel therapeutic approach. Toward this end, we have constructed and studied in vitro an adenoviral gene transfer vector that employs the promoter region of the dopamine-1 receptor to drive expression of the inward rectifier K + channel Kir2.3. The use of this neuronal promoter confers cell-type specificity and a physiological level of trans-gene expression in rat primary striatal cultures. The electrophysiological properties were confirmed in transfected human embryonic kidney cells, in which an inwardly-rectifying, Cs +-sensitive current was measured by voltage clamp. Current clamp studies of transduced striatal neurons demonstrated an increase in the firing threshold, latency to first action potential and decrease in neuronal excitability. Neurotoxin-induced activation of c-Fos, a marker of neuronal activity, was blocked in transduced neurons indicating that the decrease in electrical excitability was physiologically significant. When used in vivo, this strategy may have the potential to positively impact movement disorders by selectively changing activity of neurons belonging to the direct striatal pathway, characterized by the expression of dopamine-1 receptors.