Virus Posttranscriptional Regulatory Element Research Articles

Post-transcriptional regulatory element boosts baculovirus-mediated gene expression in vertebrate cells

Baculoviruses can express transgenes in a wide range of vertebrate cells. However, in some cells transgene expression is weak. To enhance transgene expression, we studied the effect of the Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) on baculovirus (BV)-mediated gene expression of several transgenes. A significant increase in BV-mediated gene expression was detected in several cell lines. A 10-fold increase in transgene expression was observed with the WPRE as determined by the percentage of positive cells and mean fluorescence intensity (MFI). Furthermore, a combination of optimized cell culture medium and WPRE virus led to more than a 60-fold increase in gene expression. In accordance, elevated mRNA and protein levels were detected in WPRE-virus transduced cells. In HepG2 and RaaSMC, WPRE-mediated enhancement was comparable to the previously shown positive effect of sodium butyrate on BV-mediated gene expression. Thus, inclusion of the WPRE into a baculovirus vector provides a simple means to improve BV-mediated gene expression in vertebrate cells.

275. Effect of the Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE) on Transgene Expression Controlled by the Human Synapsin 1 Gene Promoter Following Adenoviral-Mediated Gene Delivery

275. Effect of the Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE) on Transgene Expression Controlled by the Human Synapsin 1 Gene Promoter Following Adenoviral-Mediated Gene Delivery

Controlled delivery of glial cell line-derived neurotrophic factor by a single tetracycline-inducible AAV vector

An autoregulated tetracycline-inducible recombinant adeno-associated viral vector (rAAV-pTet bidiON) utilizing the rtTAM2 reverse tetracycline transactivator (rAAV-rtTAM2) was used to conditionally express the human GDNF cDNA. Doxycycline, a tetracycline analog, induced a time- and dose-dependent release of GDNF in vitro in human glioma cells infected with rAAV-rtTAM2 serotype 2 virus. Introducing the Woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) downstream to the rtTAM2 coding sequence, resulted in a more rapid induction and a higher basal expression level. In vivo, 8 weeks after a single injection of the rAAV-rtTAM2-GDNF vector encapsidated into AAV serotype 1 capsids in the rat striatum, the GDNF protein level was 60 pg/mg tissue in doxycycline-treated animals whereas in untreated animals, it was undistinguishable from the endogenous level (∼ 4 pg/mg tissue). However, a residual GDNF expression in the uninduced animals was evidenced by a sensitive immunohistochemical staining. As compared to rAAV1-rtTAM2-GDNF, the rAAV1-rtTAM2-WPRE-GDNF vector expressed a similar concentration of GDNF in the induced state (with doxycycline) but a basal level (without doxycycline) ∼ 2.5-fold higher than the endogenous striatal level. As a proof for biological activity, for both vectors, downregulation of tyrosine hydroxylase was evidenced in dopaminergic terminals of doxycycline-treated but not untreated animals. In conclusion, the rAAV1-rtTAM2 vector which expressed biologically relevant doses of GDNF in the striatum in response to doxycycline with a basal level undistinguishable from the endogenous striatal level, as measured by quantitative ELISA assay, constitutes an interesting tool for local conditional transgenesis.

176 DOXYCYCLINE-INDUCIBLE EXPRESSION OF THE hEPO GENE IN A RETROVIRUS VECTOR

The use of 'bioreactors' to address the growing demand for large quantities and increasing numbers of biopharmaceuticals is of prime strategic relevance to medical advancement. However, it is well known that constitutive overexpression of some cytokine genes may cause serious physiological disturbances in vivo and in vitro. The main objective of this study was to test the feasibility of controllable gene expression of hEPO, a glycoprotein hormone that stimulates red blood cell production. We constructed and tested retrovirus vectors designed to express the hEPO (human erythropoietin) gene under the control of tetracycline-inducible promoters. Advantages of this system over other counterparts, whose gene expressions are controlled by steroid hormones or heavy metals, are little or no cytotoxicity of the inducer and efficient turn-on/off transition. However, the most critical problem of the tetracycline-controllable gene expression system is that overall gene expression level is low. As a solution for this problem, we introduced a WPRE (woodchuck hepatitis virus post-transcriptional regulatory element) sequence into the retrovirus vector at a downstream region of either the hEPO gene or the sequence encoding rtTA (reverse tetracycline-controlled transactivator) of Tet-On system. In cooperation with tetracycline, the rtTA activates the tetracycline response promoter which controls the expression of the hEPO gene. Transformed cells derived from pig and chicken were cultured in medium supplemented with or without 1 µg mL–1 of doxycycline (a tetracycline derivative) for 48 h, and induction efficiency was measured by comparing the hEPO gene expression level using RT-PCR, Western blotting, and ELISA. Western blotting analysis showed that, among several vectors tested, the vector carrying the WPRE sequence at the 32 side of the hEPO gene was the best in both chicken and porcine cells. Quantitative analysis using ELISA resulted in a more than 10-fold increase in overall hEPO gene expression without sacrificing the efficiency of turn-on/off transition. These results should be very helpful in establishing transgenic livestock as bioreactors, making it possible to produce commercially valuable and biologically active proteins in large quantities.

Recombinant Bovine Herpesvirus 4 (BoHV-4) Expressing Glycoprotein D of BoHV-1 Is Immunogenic and Elicits Serum-Neutralizing Antibodies against BoHV-1 in a Rabbit Model

Several biological characteristics of bovine herpesvirus 4 (BoHV-4) make it a good candidate as a gene delivery vector for vaccination purposes. These characteristics include little or no pathogenicity, unlikely oncogenicity, the capability to accommodate large amounts of foreign genetic material, the ability to infect several cell types coming from different animal species, and the ability to maintain transgene expression in both undifferentiated and differentiated cells. Starting from BoHV-4 cloned as a bacterial artificial chromosome (BAC), we used MuA transposase-mediated in vitro transposition to generate recombinant BoHV-4 expressing the immunodominant glycoprotein D (gD) of BoHV-1, one of the most important pathogens of cattle. Although a cis-acting element from woodchuck hepatitis virus (the woodchuck hepatitis virus posttranscriptional regulatory element [WPRE]) in the 3' end of the gD expression cassette was required for maximal gD expression from plasmids in transient transfection assays, this element was not necessary for efficient expression of gD from recombinant BoHV-4 genomes. BoHV-4 recombinants containing gD expression cassettes with or without the WPRE expressed gD at similarly high levels. Several cell lines originating from different animal species expressed gD when infected with BoHV-4 recombinants. When rabbits were immunized with one of the recombinants, high levels of serum neutralizing antibodies against BoHV-1 were generated. This work is one of the first demonstrations of the use BoHV-4 as a vector for vaccine purposes and may provide the basis for BoHV-1 vaccination of cattle with recombinant BoHV-4.

The hepatitis B virus PRE contains a splicing regulatory element.

The posttranscriptional regulatory element (PRE) is considered to enhance hepatitis B virus (HBV) gene expression by facilitating the nuclear export of intronless viral subgenomic RNAs. Its role in the RNA metabolism of the viral pregenomic RNA (pgRNA) is currently unknown. We identified a positively cis-acting splicing regulatory element (SRE-1) and present two lines of evidence for its functionality. Firstly, in a heterologous context SRE-1 functionally substitutes for a retroviral bidirectional exonic splicing enhancer (ESE). As expected, SRE-1 is a splicing enhancer also in its natural viral sequence context, since deletion of SRE-1 reduces splicing of pgRNA in cell culture experiments. Secondly, we show that stimulation of HBV RNA splicing by the splicing factor PSF was repressed by the PRE. Analysis of a variety of PSF mutants indicated that RNA-binding and protein–protein interaction were required to enhance splicing. In addition, we show that the PRE contributed to pgRNA stability, but has little influence on its nuclear export. Herein, we report for the first time that the PRE harbors splicing stimulating and inhibiting regulatory elements controlling processing of the viral pregenome. We discuss a model in which the regulation of pgRNA splicing depends on cellular factors interacting with the PRE.

1079. Effect of WPRE on Transgene Expression Using Different Promoters in the Context of Hydrodynamically Delivered Plasmid Vectors

The Woodchuck hepatitis virus (WHV) posttranscriptional regulatory element (WPRE) facilitates nucleocytoplasmic transport of RNA mediated by several alternative pathways that may be cooperative. This element has been included in many different genetherapy vectors including lentivirus, AAV, adenovirus etc., to stimulate heterologous cDNAs expression.

773. Chemoprotection of Mouse Marrow Transplant Recipients by Ex Vivo Lentiviral Transduction of Murine tyr22 Dihydrofolate Reductase Conferring Resistance to Methotrexate

Top of pageAbstract Methotrexate (MTX) impedes tumor development by inhibiting dihydrofolate reductase (DHFR), an enzyme that catalyzes the conversion of 7,8-dihydrofolate to 5,6,7,8-tetrahydrofolate (THF). MTX depletes cells of THF, a required precursor for de novo purine and thymidine nucleotide synthesis, and has toxic effects on normal cells of the hematopoietic and gastrointestinal systems. To decrease MTX sensitivity, we introduced a MTX-resistant murine DHFR variant into mouse bone marrow by lentiviral transduction, so as to efficiently transduce non-dividing hematopoietic stem cells. We constructed HIV-1-based lentiviral vectors containing an enhanced green fluorescent protein (eGFP) gene with or without a murine tyr22 DHFR cDNA under transcriptional control of the human EF1-|[alpha]| promoter. These VSV-G-protein pseudotyped lentiviral vectors contained a self-inactivating 3|[prime]| LTR, rev response element (RRE) and woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) to enhance transduction efficiency. Viral vector was produced by co-transfection in 293T cells and concentrated 100-fold by centrifugation to a titer of 1 x 108 IU/ml, as determined by MTX-resistant colony formation and by flow cytometry for GFP expression. We transduced unfractionated bone marrow from normal Bl/6 Ly5.1 mice with either DHFR-GFP or GFP virus for 12 hours at a multiplicity of infection of 10 in the presence of cytokines (mSCF, IL-6, IL-3). Congenic Ly5.2 recipients received sub-lethal irradiation (700 cGy) prior to transplantation of 3 x 106 transduced marrow cells. MTX or PBS were administered daily starting twenty- four hours after transplantation in three recipient cohorts. Animals that received DHFR-virus transduced marrow recovered to normal hematocrit levels by week 3 in spite of MTX administration (44.7% +/- 2.5). In contrast, animals that received GFP-transduced marrow with subsequent MTX administration exhibited extremely reduced hematocrits (16.5% +/- 0.5) at week 3 and did not survive MTX dose-escalation to 2 mg/kg/day. Animals on MTX therapy that received DHFR-GFP transduced marrow exhibited increased donor cell (Ly5.1) engraftment and significant GFP marking in lymphoid (CD3, B220) and myeloid (GR-1) populations, while recipients that received GFP-transduced marrow exhibited lower levels of peripheral blood mononuclear cells and GFP+ leukocytes. Studies are currently underway to determine transgene copy number in repopulated marrow cells and assess MTX toxicity for the gastrointestinal tract. We conclude that transplantation of lentivirally transduced MTXr-DHFR transgenic marrow supports bone marrow repopulation during MTX chemotherapy in irradiated recipients, providing significant chemoprotection that permits MTX dose-escalation. These pre-clinical results support the development of a clinical gene therapy protocol to provide chemoprotection by lentiviral gene transfer.

216. Long Term Portal Vein Administration of AAV-WPRE Vector Results in Increased Incidence of Neoplastic Disease and Hepatic Pathology

Utilizing the Pahenu2 mouse model for phenylketonuria, we developed several expression vectors containing the Woodchuck Hepatitis Virus post-transcriptional regulatory element added in a rAAV-mPAH construct (rAAV-mPAH-WPRE). While these modifications resulted in increased efficacy in reducing hyperphenylalanemia, we observed severe hepatic pathology and increased incidence of hepatic and systemic neoplastic disease at the termination of these studies. Group 1 received AAV- CBmPAHdimer-WPRE with doses ranging from 1.5x109 to 4e10 IU and was sacrificed at 11 months. 44% (4/9) developed neoplastic disease that included 2 hepatocellular carcinomas, 1 hepatoma, and 1 bronchiogenic carcinoma. 6/9 (66%) had severe hepatic morphology which included myriad cytoplasmic inclusion bodies (1/9) and hepatocellular metaphase arrest. Group 2 was sacrificed at 8 months and had received a similar dose range and vector as group 1 but was ovariectomized and received either a testosterone or placebo pellet. 46% (6/13) developed the most unusual and poorly differentiated forms of neoplasia which included a peritoneal carcinosarcoma, a mesothelioma-like neoplasm of the pleura and epicardium, bilateral renal carcinoma, pulmonary and splenic lymphosarcoma, and two blastoma-like tumors with hepatic sinusoidal involvement. 7/13 (54%) also had pathologic changes in the liver as in group 1. Experimental group 3 was sacrificed at 6 months and received 4e9 AAV-CB-mPAHhd-WPRE as well as 4x109 to 2x1010 AAV-CB-RzI209 vector. None developed neoplastic disease but 60% (3/5) did develop altered hepatocellular morphology as above. Mice from group 4 were sacrificed at 6 and 12 months and received between 4e9 and 3e10 IU of AAV-CB-mPAHhrd+RzI209 (no WPRE). None of these mice (0/8) developed neoplastic disease, but 37.5% (3/8) developed mild to moderate changes in hepatocellular morphology that were consistent with increased metabolic activity. The woodchuck hepatitis virus (WHV) contains a highly conserved 154 amino acid HBV X protein (HBx) that is implicated as a key element in the development of hepatocellular carcinoma. HBx is a trans- activating protein that is often expressed in hepatocellular carcinoma (HCC) and surrounding nontumor liver tissue. The WPRE element we used included an amino terminal 60 amino acid fragment of the HBx protein (1503-1680). To determine if vector was present in neoplastic tissue, and more specifically to look for the presence of HBx, PCR (TAQMan|[copy]|) was employed using HBx probes. HBx DNA was not detected in tumoral tissue although it was present in transduced liver. Immunohistochemical detection of Hbx antigen was observed on the periphery of 1 liver tumor and scattered randomly throughout an intestinal tumor. These results imply that AAV-vector is not integrated into the tumor, but rather it is possible the Hbx element is acting in a |[ldquo]|hit and run|[rdquo]| fashion when administered systemically and may activate cellular oncogenes at random.

929. Functional Potential of Human T Cells Following Lentiviral Suicide Gene Transduction

Top of pageAbstract Clinical trials of retroviral T cell suicide gene therapy following allogeneic haematopoietic stem cell transplantation (HSCT) have shown that T cells causing graft versus host disease (GVHD) can be eliminated following the administration of the prodrug Ganciclovir (GCV). This strategy offers the possibility of harnessing powerful donor lymphocyte immunity in patients undergoing alternative donor HSCT (in both the HLA matched or mismatched settings) who would otherwise be at high risk of intractable GVHD. A number of limitations have been identified, including the detection of GCV resistant alternate splice variants, evidence of impaired anti-viral immunity, and immune response against gene-modified cells. We have generated lentiviral vector systems capable of mediating gene transfer to T cells in the absence of T cell receptor or CD3 activation. The HIV-1 based, self-inactivating constructs encode a central polypurine tract (cPPT) element and an X-protein start site mutated woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). A dual suicide gene/selection marker encoding an enhanced HSVTK mutant (with corrected splice site) and truncated CD34 (tCD34) has been incorporated. Virus was pseudotyped with the Vesicular Stomatitis Virus (VSV-G) or a chimaeric RD114 envelope encoding the Molony leukaemia virus cytoplasmic tail . We compared transduction protocols using either full activation of T cells (anti-CD3/CD28 conjugated beads plus Interleukin-2) or pre-treatment of T cells with IL-2 or IL-7 cytokines alone or in combination. We show that the culture of T cells in media containing cytokines for 96h prior to exposure to viral supernatant renders cells permissive to lentiviral transduction. Transuction efficiency in IL-2 or IL-7 averaged 25% comapred to over 70% for fully activated cells.We observed reduced cell division on the basis of CFSE dye dilution in the cytokine stimulated cells, and flow cytometry confirms greater retention of CD45RA+ naive populations, with less skewing towards the CD45RO+CD27+ subgroups. T cells transduced in the presence of IL-7 retained the highest levels of viability. Preservation of virus specific responses was confirmed by tracking CMV specific T cells through the transduction procedure using tetramer staining. After enrichment on the basis of CD34 expression, transduced cells mediated intact responses against autologous dendritic cells pulsed with CMV. Similarly, proliferative responses against allogeneic dendritic cells were best preserved following IL-7 stimulation. Primary T cells, and virus specific T cells, specific for Adenovirus or CMV, were readily eliminated by exposure to GCV or the less toxic agent Aciclovir and there was no evidence splice variants resistant to prodrug action. The vectors offer the potential of high efficiency transduction and selection, efficient prodrug mediated elimination with preservation of T cell effector responses using cytokine pre-stimulation.

808. Optimizing Non-Primate Lentiviral Vectors for Gene Transfer of CFTR to Airway Epithelia

Suboptimal expression from Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) cDNA delivered by viral vectors has, in part, limited the use of gene transfer as a therapy for cystic fibrosis. Previously we described a lentiviral vector developed from feline immunodeficiency virus (FIV) pseudotyped with baculovirus GP64 which transduces nasal epithelia in mice and persistently expresses a reporter gene. The goal of the current project is to further improve transgene expression levels through several modifications to this FIV vector, and to test the optimized vector's effects on protein expression. Modifications include rendering the lentiviral vector |[ldquo]|self-inactivating|[rdquo]| by deleting the U3 region of the 3’ LTR, thus providing a layer of safety. The FIV central polypurine tract (cPPT) element was inserted upstream of the internal promoter and the woodchuck hepatitis virus posttranscriptional regulatory element (wPRE) was incorporated downstream of the reporter gene. Also, two sites were mutated, a major splice donor and the start codon of the partial Gag sequence, preventing production of undesired product. In addition, a newly identified post- transcriptional control element from the 5’ LTR of spleen necrosis virus (SNVU5) was incorporated into the delivery vector. The Rous sarcoma virus promoter and the reporter gene, firefly luciferase, were used to test relative expression of the intermediate vectors in the production of the optimized FIV vector. Expression was quantified in two model systems transduced with modified FIV lentiviral vector: cultured HT1080 cells and Balb/c mice. As determined by luciferase activity assay, in vitro transductions with intermediate vectors showed an enhanced luciferase expression with wPRE and SNVU5, but no enhanced expression with the addition of cPPT. As determined by CCD camera detection of bioluminescence in vivo, both the wPRE and SNVU5 elements significantly increased luciferase expression in the FIV lentiviral vector, but the cPPT did not. The optimized and intermediate vectors were used to deliver the CFTR gene, and expression was detected by real-time PCR targeting CFTR mRNA. The optimized vector produced approximately 20-fold increase in CFTR mRNA as compared with the control vector. Vector constructs with epithelial specific promoters will be used to transduce airway epithelia in vitro and in vivo, and the contribution of the promoter to expression levels, persistence, and cell specific expression will be assessed. The long-term goal is to use an optimized viral vector construct to express sufficient CFTR protein in the airways of humans with cystic fibrosis to correct the chloride ion transport defect.

339. FIV Lentiviral Vector Gene Transfer for Hemophilia A

Hemophilia A is an X-linked genetic disorder resulting in deficiencies in coagulation Factor VIII and a risk of sustained bleeding after trauma or injury. Hemophilia A is an attractive candidate for treatment by gene transfer because only partial correction is required for clinically relevant effects. We previously reported that gene transfer to the liver of E16 FVIII deficient mice using a lentiviral vector derived from feline immunodeficiency virus (FIV) resulted in sustained partial correction of the bleeding disorder. We are now investigating the impact of several vector and transgene modifications on gene transfer to hepatocytes using baculovirus GP64 pseudotyped FIV. The goals of the project are to improve transgene expression through several vector modifications and to test the optimized vector's effects on protein expression. Modifications include rendering the lentiviral vector |[ldquo]|self-inactivating|[rdquo]| by deleting the U3 region of the 3’ LTR, inserting the FIV central polypurine tract (cPPT) element upstream of the internal promoter, and incorporating the woodchuck hepatitis virus posttranscriptional regulatory element (wPRE) downstream of a luciferase reporter gene or the FVIII cDNA. Furthermore, two sites were mutated, a major splice donor and the start codon of the partial Gag sequence, preventing production of undesired product. In addition, the post- transcriptional control element from the 5’ LTR of spleen necrosis virus (SNVU5) was incorporated into the delivery vector. The liver- specific mouse albumin enhancer and human alpha-1-antitrypsin promoter (Alb/HAAT) are being used to direct firefly luciferase, B- domain deleted human FVIII (BDD-FVIII), and a FVIII cDNA engineered to include several asparagine-linked oligosaccharides within a short B-domain spacer (F226aa/N6) in wild type and E16 FVIII deficient mice on a congenic C57/BL6 background. Findings from these ongoing experiments will be reported.

Woodchuck hepatitis virus post-transcriptional regulatory element deleted from X protein and promoter sequences enhances retroviral vector titer and expression

Introduction of the post-transcriptional regulatory element (PRE) of woodchuck hepatitis virus (WHV) into the 3' untranslated region of retroviral and lentiviral gene transfer vectors enhances both titer and transgene expression. Optimal use of the PRE is often necessary to obtain vectors with sufficient performance for therapeutic applications. The enhancing activity of the PRE depends on the precise configuration of its sequence and the context of the vector and cell into which it is introduced. However, data obtained in the context of WHV-associated hepatocellular carcinomas suggests that the PRE might potentially contribute to tumorigenesis, especially if encoding a truncated version of the WHV X protein. Oncogenic side effects of lentiviral vectors containing the PRE have reinforced these safety concerns, although a causal role of the PRE remained unproven. Here, we demonstrate that PRE mutants can be generated that are devoid of X protein open reading frames (ORFs) as well as other ORFs exceeding 25 amino acids, without significant loss of RNA enhancement activity. Furthermore, the X protein promoter could be deleted without compromising the enhancement of vector titers and transgene expression. Such a modified PRE sequence appears useful for future vector design.

Rapid Generation of Stable Transgenic Embryonic Stem Cell Lines Using Modular Lentivectors

Generation of stable transgenic embryonic stem (ES) cell lines by classic transfection is still a difficult task, requiring time-consuming clonal selection, and hampered by clonal artifacts and gene silencing. Here we describe a novel system that allows construction of lentivectors and generation of stable ES cell lines with > 99% transgene expression within a very short time frame. Rapid insertion of promoters and genes of interest is obtained through a modular recombinational cloning system. Vectors contain central polypurine tract from HIV-1 element and woodchuck hepatitis virus post-transcriptional regulatory element as well as antibiotic resistance to achieve optimal and homogenous transgene expression. We show that the system 1) is functional in mouse and human ES cells, 2) allows the generation of ES cells expressing genes of interest under the control of ubiquitous or tissue-specific promoters, and 3) allows ES cells expressing two constructs through selection with different antibiotics to be obtained. The technology described herein should become a useful tool in stem cell research.

Lentiviral transduction of human hematopoietic cells by HIV‐1‐ and SIV‐based vectors containing a bicistronic cassette driven by various internal promoters

Lentiviral gene transfer into hematopoietic cells has been mostly optimized with vectors carrying a single reporter gene. For many clinical applications, lentiviral vectors should contain more than one gene because transduced cells should be enriched by a selectable marker or killed for safety reasons after use. Thus, we compared various vectors containing a bicistronic cassette driven by different ubiquitous promoters for their ability to transduce human T-lymphocytes, CD34+-cells, and dendritic cells (DCs) derived from CD34+-cells or monocytes. We designed HIV or SIV constructs containing a bicistronic cassette composed of two reporter genes (thy1/GFP) linked by an internal ribosome entry site sequence and driven by the cytomegalovirus (CMV) or elongation factor 1alpha (EF1alpha) promoters. The woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) was or not inserted within the constructs, the Vpx accessory protein was or not used for SIV vectors. Target cells were infected at the same multiplicity of infection, transduction efficiency was analyzed both by flow cytometry and vector integration. For T-cells, HIV-based vectors/WPRE+ in which the thy1/GFP cassette was driven by the EF1alpha promoter were more efficient than SIV-based vectors. For CD34+-cells and CD34+-derived DCs, better thy1/GFP expression was achieved when the CMV promoter drove the cassette inserted into HIV-based vectors/WPRE+. Conversely, for monocyte-derived DCs, the cassette yielded better thy1/GFP expression when inserted into SIV-based vectors/WPRE+ and driven by the CMV or EF1alpha promoters, the use of Vpx significantly improving the expression levels. Our results provide guidelines for improving the transduction of T-cells, CD34+-cells or DCs with lentiviral bicistronic vectors designed for clinical applications.

620. SMC-Specific Transcription Elements for Improved Adenoviral-Mediated Transgene Expression in Saphenous Veins

Top of pageAbstract Coronary artery bypass grafting (CABG) using autologous saphenous veins (SV) is an essential procedure to restore blood supply to the heart. However, long-term patency rates remain poor (93%, 74% and 41% at 1, 5 and 10 years post-grafting1) as vascular damage and remodeling often leads to neointima formation and accelerated atherosclerosis. Pharmacological interventions are largely ineffective and since the saphenous vein is accessible for ex vivo modification prior to arterial grafting, this presents an ideal window of opportunity for gene therapy. Several candidate therapeutic genes predicted to counteract vein graft failure have shown success pre-clinically but clinical translation is yet to take place. One of the most important issues is optimisation of transgene expression locally in the vein graft following gene delivery. We therefore assessed the ability of first-generation adenoviral vectors harbouring different promoter and post-transcriptional regulatory elements (Figure 1, ref. 2) for their expression levels in saphenous vein cells in vitro, ex vivo and in vivo. Our results demonstrate that Ad-PREP [in which lacZ expression is driven by the murine CMV promoter (mCMV), the Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) and a fragment of the smooth muscle myosin heavy chain promoter (RE)] produces significantly higher lacZ expression levels in primary saphenous vein smooth muscle cells (20-fold vs. RAd35 at m.o.i. 250) and endothelial cells (8-fold), but not in hepatocytes (1.23-fold), compared to RAd35, a standard Ad5-hCMV-bgal vector. This was particularly evident after short virus:cell incubation times (of 30 minutes), clearly relevant to the clinical setting. High levels of lacZ expression were also achieved with this vector following infection of human saphenous vein segments ex vivo, and 7 days after grafting autologous saphenous veins into pig carotid arteries in vivo. These results demonstrate that high transgene expression levels in saphenous vein can be achieved by the mCMV promoter, in combination with smooth muscle cell-specific and post-transcriptional regulatory elements.

Enhancing rAAV vector expression in the lung

Despite favorable DNA transfer efficiency, gene expression from recombinant adeno-associated virus (rAAV2) vectors in the lung has been variable in the context of cystic fibrosis (CF) gene therapy. This is due, in part, to the large size of the CF transmembrane regulator (CFTR)-coding sequence which necessitates the use of compact endogenous promoter elements versus stronger exogenous promoters. We evaluated the possibility that gene expression from rAAV could be improved by using AAV capsid serotypes with greater tropism for the apical surface of airway cells (i.e. rAAV5 or rAAV1) and/or using strong promoters such as the cytomegalovirus (CMV) enhancer/chicken beta-actin hybrid (Cbeta) promoter. The relative activity of the CMV immediate-early (CMVie) promoter, the Cbeta promoter, and the Cbeta promoter with a downstream woodchuck hepatitis virus post-transcriptional regulatory element (wpre) were assessed in vitro and in vivo in C57\Bl6 mice using human alpha-1 antitrypsin (hAAT) as a secreted reporter. In vivo, the Cbeta-AAT-wpre group achieved maximum serum levels of 1.5 mg/ml of hAAT. AAV capsid serotypes were then compared in vivo utilizing the transcriptionally optimized CB-wpre cassette in rAAV serotype 1, 2 or 5 capsids (rAAV1, rAAV2, and rAAV5), utilizing luciferase as a reporter to compare expression over a wide dynamic range. The pulmonary luciferase levels at 8 weeks were similar in rAAV5 and rAAV1 groups (2.9 x 10(6) relative light units (RLU)/g tissue and 2.7 x 10(6) RLU/g tissue, respectively), both of which were much higher than rAAV2. Although the advantage of rAAV5 over rAAV2 in the lung has already been described, the availability of another serotype (rAAV1) capable of efficient gene transfer in the lung could be useful.

224 TETRACYCLINE-INDUCIBLE GENE EXPRESSION WITH RETROVIRUS VECTOR

One of the critical problems to be solved in transgenic animal production is non-controllable constitutive expression of the foreign gene, which usually results in serious physiological disturbances in the transgenic animal. To circumvent this problem, we constructed and tested two retrovirus vectors designed to express the GFP (green fluorescent protein) gene under the control of tetracycline-inducible promoters. To maximize the GFP gene expression at turn-on state, the WPRE (woodchuck hepatitis virus post-transcriptional regulatory element) sequence was also introduced into the retrovirus vector downstream of either the GFP gene or the sequence encoding rtTA (reverse tetracycline-controlled transactivator). Transformed cells derived from porcine fetus were cultured in the medium supplemented with or without doxycycline (tetracycline derivative) for 48 h, and induction efficiency was measured by comparing the GFP gene expression level using fluorometry and western blotting. Higher GFP expression was observed from the vector carrying the WPRE sequence at the 3′ side of the GFP gene, while tighter expression control (up to 20-fold) was obtained from the vector in which the WPRE sequence was placed at the 3′ side of the rtTA sequence. Encouraged with these data, we substituted the hPTH (human parathyroid hormone) gene for the GFP gene in the retrovirus vector. The porcine fetal fibroblast cells transformed by the modified retrovirus vector secreted hPTH into the medium under the tight control of doxycycline as observed in GFP expression. The resulting porcine cells secreting hPTH will be used in nuclear transfer experiment. This study was financially supported by the National Livestock Research Institute RDA (Suwon 441-350, Korea), ARPC (Agriculture R & D Promotion center, 2002–2005), and by grant No. R11-2002-100-01000-0 from the ERC program of the Korea Science & Engineering Foundation.

Lentiviral Delivery of Suicide Gene and Selection Molecules to Non-Dividng T Cells.

T cell suicide gene therapy is designed to permit the use of donor T cells for their powerful anti-leukaemic and anti-viral effects following haematopoietic stem cell transplantation. In the event of graft versus host disease (GVHD) such cells can be eliminated through the administration of suicide-gene dependent prodrugs. Though the feasibility of the strategy has been demonstrated in clinical trials, there has been a lower incidence of GVHD than anticipated and evidence of impaired anti-viral immunity. Current vectors under clinical investigation are based on retroviral delivery systems and require T cells to be actively dividing at the time of vector exposure to for successful transduction. The process of ex-vivo activation to induce mitogenesis has been shown to impair subsequent functional potential, and even under optimized conditions using combinations of anti-CD3 and anti-CD28 antibodies, T cells become prone to exhaustion. It has been previously reported that lentiviral vectors permit T cell transduction without the need for mitogenic stimulation if used in combination with certain cytokines such as Interleukin-2 (IL-2) or Interleukin-7 (IL-7). Thus we have adapted a self-inactivating lentiviral vector system based on HIV-1 and encoding strong promoter elements derived from the long terminal repeat (LTR) regions of Spleen Focus forming virus (SFFV) for the delivery of suicide genes to T cells. The Woodchuck hepatitis virus post-transcriptional regulatory element and the central polypurine tract element of HIV-1 have been incorporated to enhance transduction efficiency and increase transgene expression. T cells cultured in the presence of either IL-2 or IL-7 alone, or in combination could be transduced at between 20–30% efficiency following a single round of exposure to virus pseudotyped with the vesicular stomatitis virus envelope or the feline endogenous leukaemia virus envelope. By the end of the procedure there were minimal changes in T cell surface phenotype, preservation of niave/memory subsets and retention of virus specific populations as quantified by tetramer staining of Cytomegalovirus (CMV) specific T cells. Functional analysis indicated preservation of proliferative responses to autologous dendritic cells pulsed with CMV antigen. In addition, strong responses in mixed lymphocyte culture indicated intact allo-reactive responses. Cells transduced to express a fusion construct encoding a variant herpes simplex thymidine kinase fused to the truncated CD34 selection marker could be enriched to >95% purity by a single round of anti-CD34 magnetic bead selection. The functional integrity of the suicide gene was confirmed by elimination of enriched cells following exposure to the prodrugs Gancilcovir and Aciclovir. In conclusion, non-dividing T cells transduced with suicide gene/selection transgenes using lentiviral constructs, retain phenotypic characteristics and functional responsiveness.

The hybrid cytomegalovirus enhancer/chicken beta-actin promoter along with woodchuck hepatitis virus posttranscriptional regulatory element enhances the protective efficacy of DNA vaccines.

DNA vaccines represent a novel and powerful alternative to conventional vaccine approaches. They are extremely stable and can be produced en masse at low cost; more importantly, DNA vaccines against emerging pathogens or bioterrorism threats can be quickly constructed based solely upon the pathogen's genetic code. The main drawback of DNA vaccines is that they often induce lower immune responses than traditional vaccines, particularly in nonrodent species. Thus, improving the efficacy of DNA vaccines is a critical issue in vaccine development. In this study we have enhanced the efficacy of DNA vaccines by adopting strategies that increase gene expression. We generated influenza-hemagglutinin (HA)-encoding DNA vaccines that contain the hybrid CMV enhancer/chicken beta-actin (CAG) promoter and/or the mRNA-stabilizing post-transcriptional regulatory element from the woodchuck hepatitis virus (WPRE). Mice were immunized with these DNA vaccines, and the influenza-HA-specific cellular and humoral immune responses were compared with a conventional, HA-encoding DNA vaccine whose gene expression was driven by the CMV immediate-early promoter (pCMV-HA). CAG promoter-driven DNA vaccines elicited significantly higher humoral and cellular immune responses compared with the pCMV-HA vaccine. DNA vaccines consisting of both CAG and WPRE elements (pCAG-HA-WPRE) induced the highest level of protective immunity, such that immunization with 10-fold lower DNA doses prevented death in 100% of the mice upon lethal viral challenge, whereas all mice immunized with the conventional pCMV-HA vaccine succumbed to influenza infection.