Glucocorticoid-inducible gene expression vectors for use in Drosophila melanogaster Schneider 2 cells

Inducible, vector-based, expression systems that allow fine control of transgene expression are gaining more and more use in fundamental research as well as in therapeutic applications. In an effort to develop a tightly regulated heterologous expression system for Drosophila Schneider 2 cells, three different inducible reporter constructs were compared. These comprised six copies of the glucocorticoid response element fused to one of three distinct types of Drosophila gene promoters: (1) a TATA-box containing, (2) a TATA-less and (3) a bidirectional core sequence. These were fused to a luciferase reporter gene. The promoter constructs displayed different basal as well as agonist-induced activities. The implications of the observations made are discussed in the context of promoter properties and of induction of genes that may be studied in Drosophila.     

Modulation of neuronal survival and axonal growth in vivo by tetracycline-regulated neurotrophin expression.

Vector systems for the regulated and reversible expression of therapeutic genes are likely to improve the safety and efficacy of gene therapy for medical disease. In the present study, we investigated whether the expression of genes transferred into the central nervous system by ex vivo gene therapy can be regulated in vivo leading to controlled neuronal survival and axonal growth. Primary rat fibroblasts were transfected with a retrovirus containing a tetracycline responsive promoter for the expression of the neurotrophin nerve growth factor (NGF) or green fluorescent protein as a control (GFP). After lesions of basal forebrain cholinergic neurons, NGF-mediated neuronal rescue and axonal growth could be completely controlled over a 2-week period by the addition or removal of the tetracycline modulator doxycycline in the animals' drinking water. Further, continued expression of the reporter gene GFP could be reliably and repeatedly turned on and off in the injured CNS for at least 3 months post-grafting, the longest time point investigated. These data constitute the first report of regulated neuronal rescue and axonal growth by controlled neurotrophin gene delivery and long-term, regulated expression using ex vivo CNS gene therapy.     

Increased persistence of lung gene expression using plasmids containing the ubiquitin C or elongation factor 1alpha promoter.

For effective gene therapy of chronic disease, persistent transgene expression at therapeutic levels is required. Clinical studies of airway gene transfer in patients with cystic fibrosis (CF) have resulted in short-lived transgene expression. We used intra-nasal dosing of naked plasmid DNA to the murine lung as a model for investigating the duration of airway gene transfer from a series of reporter expression plasmids. Transgene expression was transient when mediated by the viral promoters CMV, RSV and SV40, falling to less than 10% of peak expression after 2 weeks, although the presence of the adenoviral E4ORF3 gene in cis, resulted in extended duration of reporter activity from the CMV promoter. Transient expression from these promoters was not due to loss of the vector as determined by quantitative TaqMan PCR analysis. However, use of the promoters from the human polybiquitin C (UbC) and the elongation factor 1alpha (EF1alpha) genes resulted in persistent gene expression in the mouse lung. The UbC promoter directed high-level reporter activity which was maintained for up to 8 weeks and was still detectable 6 months after a single administration. Such persistent airway transgene expression from a nonviral vector without the concomitant expression of a potential antigen has not been reported previously. Thus, despite the persistence of vector DNA in vivo, attenuation of promoter function may lead to silencing of transgene expression and careful selection of promoter sequences is recommended for in vivo gene transfer.     

Direct correlation between positron emission tomographic images of two reporter genes delivered by two distinct adenoviral vectors

Biodistribution, magnitude and duration of a therapeutic transgene's expression may be assessed by linking it to the expression of a positron emission tomography (PET) reporter gene (PRG) and then imaging the PRG's expression by a PET reporter probe (PRP) in living animals. We validate the simple approach of co-administering two distinct but otherwise identical adenoviruses, one expressing a therapeutic transgene and the other expressing the PRG, to track the therapeutic gene's expression. Two PET reporter genes, a mutant herpes simplex virus type 1 thymidine kinase (HSV1-sr39tk) and dopamine-2 receptor (D(2)R), each regulated by the same cytomegalovirus (CMV) promoter, have been inserted into separate adenoviral vectors (Ad). We demonstrate that cells co-infected with equivalent titers of Ad-CMV-HSV1-sr39tk and Ad-CMV-D(2)R express both reporter genes with good correlation (r(2) = 0.93). Similarly, a high correlation (r(2) = 0.97) was observed between the expression of both PRGs in the livers of mice co-infected via tail-vein injection with equivalent titers of these two adenoviruses. Finally, microPET imaging of HSV1-sr39tk and D(2)R expression with 9-(4-[(18)F]fluoro-3-hydroxymethylbutyl) guanine ([(18)F]FHBG) and 3-(2-[(18)F]fluoroethyl)spiperone ([(18)F]FESP), utilizing several adenovirus-mediated delivery routes, illustrates the feasibility of evaluating relative levels of transgene expression in living animals, using this approach.     

Efficient gene transfer and long-term expression in neurons using a recombinant adenovirus with a neuron-specific promoter

Adenoviruses are highly efficient vectors for gene transfer into brain cells. Restricting transgene expression to specific cell types and maintaining long-term expression are major goals for gene therapy in the central nervous system. We targeted gene expression to neurons by constructing an adenoviral vector that expressed the E. coli LacZ reporter gene under the control of the rat neuron-specific enolase promoter (Ad-NSE). Expression from Ad-NSE was compared with that from an adenoviral vector encoding the same reporter gene under the control of the Rous sarcoma virus LTR promoter (Ad-RSV). Both recombinant adenoviruses were injected stereotactically into rat hippocampus, cerebellum and striatum. Anatomical and immunohistochemical analyses of the Ad-NSE-stained cells showed that neurons were preferentially transduced. More neurons were stained in the hippocampus following infection with Ad-NSE than with Ad-RSV. Cytotoxicity from Ad-NSE was lower than from Ad-RSV. beta-Galactosidase gene expression after Ad-NSE infection remained stable for 3(1/2) months, and was detectable for 6 months. Thus, the NSE-adenoviral vector can be used to transfer potentially therapeutic genes into neuronal cells. The use of a cell-specific promoter also resulted in high in vivo efficiency and long-term transgene expression.     

Development of a transposon-based approach for identifying novel transgene insertion sites within the replicating adenovirus.

Therapeutic gene delivery from an oncolytic adenovirus (Ad) is one approach to enhancing the potency of Ad-based virotherapies for cancer. To identify therapeutic transgene insertion sites compatible with the replicating virus, a methodology that broadly scans the viral genome is needed. To address this we modified a transposon (Tn7)-based in vitro transposition system to take advantage of its nonprejudiced scanning ability to identify insertion sites compatible with viral replication. Using this system with a plasmid containing an E3-deleted Ad5, we identified several unique sites for promoter-based expression cassette insertions within the Ad genome. The transposon-based expression cassette is bounded by PmeI restriction endonuclease sites unique to the transposon, making expression cassette substitutions easy to perform. Additional expression cassettes containing different promoters and reporter genes were substituted into two of the newly identified transgene insertion sites. The results suggest that the ease and orientation of expression cassette substitution depend on both the insertion site location and the promoter and gene of the replacement expression cassette. These studies establish the transposon-based system as an efficient approach to scanning the Ad genome and identifying insertion sites compatible with viral replication and represents a powerful tool for the development of armed therapeutic viruses for cancer.     

A novel system for mitigation of ectopic transgene expression induced by adenoviral vectors

Adenoviral (Ad) vectors are good candidates for gene therapy in view of their high in vivo gene delivery efficiency. However, greater control over the tissue distribution of transgene expression is required to avoid potentially deleterious effects in non-target organs. In this regard, the liver is particularly at risk due to the high natural tropism of Ad for this organ, where dose limiting toxicity has been seen due to toxic transgene expression. We hypothesized that the cre/loxP system could be utilized to reduce unintended transgene expression at this site. This concept was tested using an Ad vector (AdLCLLL) carrying a reporter gene cassette in which the promoter and luciferase gene were flanked by LoxP sequences. Co-administration of this vector with a second vector carrying the cre recombinase gene in vitro and in vivo resulted in specific down-regulation of transgene expression. This novel approach thus has the potential to improve the safety of gene therapy strategies that rely upon the delivery of genes which may be hepatotoxic.     

Inducible long-term gene expression in brain with adeno-associated virus gene transfer

Recombinant adeno-associated virus (rAAV) vectors hold promise for treating a number of neurological disorders due to the ability to deliver long-term gene expression without toxicity or immune response. Critical to these endeavors will be controlled expression of the therapeutic gene in target cells. We have constructed and tested a dual cassette rAAV vector carrying a reporter gene under the control of the tetracycline-responsive system and the tetracycline transactivator. Transduction in vitro resulted in stable expression from the vector that can be suppressed 20-fold by tetracycline treatment. In vivo experiments, carried out to 6 weeks, demonstrated that vector-transduced expression is sustained until doxycycline administration upon which reporter gene expression is reduced. Moreover, the suppression of vector-driven expression can be reversed by removal of the drug. These studies demonstrate long-term regulated gene expression from rAAV vectors. This system will provide a valuable approach for controlling vector gene expression both in vitro and in vivo.     

A single lentivirus vector mediates doxycycline-regulated expression of transgenes in the brain

A single lentivirus vector allowing doxycycline-regulated expression of transgenes in the brain was generated by incorporating the tetracycline (Tet)-dependent regulatory system into the backbone of the vector. Two distinct expression cassettes were inserted upstream and downstream from the central Flap sequence that provides for enhanced transduction of nondividing cells. The first cassette was used to express the transgene under the control of the Tet-dependent minimal cytomegalovirus promoter. The second cassette was employed to express constitutively the Tet-dependent transactivator rtTA2-M2, which activates the Tet-dependent promoter after binding of doxycycline (Tet-on system). Vectors carrying luciferase and tyrosine hydroxylase as the transgene were constructed, tested in astroglia-rich primary cultures, and injected into the striata of rats. The constructs allowed in vitro and in vivo robust expression of the transgene that could be regulated over two orders of magnitude by the addition and withdrawal of doxycycline. The vector may thus be useful for many applications in gene therapy research, including the development of a therapeutic protocol for the treatment of Parkinson's disease based on the restoration of regulated dopamine production.     

Direct comparison of hepatocyte-specific expression cassettes following adenoviral and nonviral hydrodynamic gene transfer

Hepatocytes are a key target for treatment of inborn errors of metabolism, dyslipidemia and coagulation disorders. The development of potent expression cassettes is a critical target to improve the therapeutic index of gene transfer vectors. Here we evaluated 22 hepatocyte-specific expression cassettes containing a human apo A-I transgene following hydrodynamic transfer of plasmids or adenoviral transfer with E1E3E4-deleted vectors in C57BL/6 mice. The DC172 promoter consisting of a 890 bp human alpha(1)-antitrypsin promoter and two copies of the 160 bp alpha(1)-microglobulin enhancer results in superior expression levels compared to constructs containing the 1.5 kb human alpha(1)-antitrypsin promoter, the 790 bp synthetic liver-specific promoter or the DC190 promoter containing a 520 bp human albumin promoter and two copies of the 99 bp prothrombin enhancer. The most potent expression cassette consists of the DC172 promoter upstream of the transgene and two copies of the hepatic control region-1. Minicircles containing this expression cassette induce persistent physiological human apo A-I or human factor IX levels after hydrodynamic transfer. In conclusion, in this comparative study of 22 hepatocyte-specific expression cassettes, the DC172 promoter in combination with two copies of the hepatic control region-1 induces the highest expression levels following hydrodynamic and adenoviral transfer.     

Suicidal gene therapy in an NF-κB-controlled tumor environment as monitored by a secreted blood reporter

The nuclear factor-κB (NF-κB) is known to be activated in many cancer types including lung, ovarian, astrocytomas, melanoma, prostate as well as glioblastoma, and has been shown to correlate with disease progression. We have cloned a novel NF-κB-based reporter system (five tandem repeats of NF-κB responsive genomic element (NF; 14?bp each)) to drive the expression cassette for both a fusion between the yeast cytosine deaminase and uracil phosphoribosyltransferase (CU) as a therapeutic gene and the secreted Gaussia luciferase (Gluc) as a blood reporter, separated by an internal ribosomal entry site (NF-CU-IGluc). We showed that malignant tumor cells have high expression of Gluc, which correlates to high activation of NF-κB. When NF-κB was further activated by tumor necrosis factor-α in these cells, we observed up to 10-fold increase in Gluc levels and therefore transgene expression in human glioma cells served to greatly enhance the sensitization of these cells to the prodrug, 5-fluorocytosine both in cultured cells and in vivo subcutaneous tumor xenograft model. This inducible system provides a tool to enhance the expression of imaging and therapeutic genes for cancer therapy.     

Lentivirus vector-mediated expression of tumor-associated epitopes by human antigen presenting cells

Directing the human immune system to recognize and eliminate tumor cells is the ultimate goal of cancer immunotherapy. Vaccinating patients with autologous antigen presenting cells (APC) expressing tumor-associated antigens (TAA) represents a promising approach for activating tumor-reactive T cells in vivo. In addition, APC expressing TAA provide a means of generating tumor-specific T cells in vitro, for therapeutic and diagnostic applications. Lentiviral vectors are attractive vehicles for introducing TAA-encoding genes into APC. In this study, lentiviral vectors expressing the reporter gene GFP or the melanoma-associated antigen tyrosinase were used to transduce three different kinds of human APC: monocyte-derived dendritic cells (DC), CD40L-activated B lymphocytes, and Epstein Barr virus (EBV)-transformed B lymphocytes. Using optimized transduction conditions for each cell type, tyrosinase was expressed at levels sufficient to stimulate antigen-specific major histocompatibility complex (MHC) class I-restricted T cells from melanoma patients. While transduced EBV-B cells demonstrated the highest level of transgene expression, optimal T-cell recognition was achieved with transduced DC. Substituting the CAG promoter for PGK in lentiviral constructs enhanced transgene expression in DC and EBV-B cells, amplifying T cell recognition. Lentiviruses inducing sustained transgene expression with relatively low cellular toxicity and background viral gene expression may be ideal vectors for immunotherapeutic applications.     

Spatial and temporal control of transgene expression through ultrasound-mediated induction of the heat shock protein 70B promoter in vivo

Adenoviral vectors have been constructed that express the transgenes luciferase (Adeno-HSP-Luc) or Fas ligand (Adeno-HSP-FasL) under the control of the heat shock protein 70B (hsp70B) promoter. Cultures infected with Adeno-HSP-Luc transiently expressed high levels of luciferase after heat shock. When cultures infected with Adeno-HSP-FasL were maintained at 37 degrees C, no transgene expression was observed, but when cultures were exposed to heat stress, transgene expression resulted in apoptotic cell death. In vivo, transgene expression was induced by ultrasound-mediated heating of adenovirus-infected tissue. In mice or rats injected with the Adeno-HSP-Luc construct, high levels of localized expression of luciferase activity were observed in regions subjected to ultrasound-mediated irradiation. Adeno-HSP-FasL was administered systemically to mice via the tail vein to evaluate safety. Animals receiving Adeno-HSP-FasL in the absence of ultrasound treatment did not display liver toxicity, whereas animals receiving ultrasound treatment to induce the expression of Fas ligand from the hsp70B promoter had significant increases in serum levels of liver enzymes. These data demonstrate that combining the inducible hsp70B promoter with ultrasound induction allows safe local expression of cytotoxic genes with possible therapeutic utility.     

Noninvasive Imaging of Cardiac Gene Expression and Its Future Implications for Molecular Therapy

Innovative approaches for cardiovascular molecular therapy are rapidly evolving, and translational efforts from experimental to clinical application are increasing. Gene and cell therapy hold promise for treatment of heart disease, but despite progress, some basic principles are still under development. Open issues are, e.g., related to the optimal method for delivery, to therapeutic efficacy, to time course and magnitude of gene expression, and to the fate of transplanted cells in target and remote areas. The use of reporter genes and labeled reporter probes for noninvasive imaging provides the methodology to address these questions by assessment of location, magnitude, and persistence of transgene expression in the heart and the whole body. Coexpression of a reporter gene allows for indirect imaging of the expression of a therapeutic gene of choice. Furthermore, reporter genes can be transferred to stem cells prior to transplantation for serial monitoring of cell viability using gene product imaging. Additionally, functional effects of therapy on the tissue level can be identified using established imaging approaches to determine blood flow, metabolism, innervation, or cell death. Measures of transgene expression can then be linked to physiologic effects and will refine the understanding of basic therapeutic mechanisms. Noninvasive gene-targeted imaging will thus enhance the determination of therapeutic effects in cardiovascular molecular therapy in the future.     

Hair follicle stem cell-targeted gene transfer and reconstitution system

Gene transfer to hair follicle (HF) epithelium is an attractive approach for not only treating skin diseases, but also many systemic disorders. In this study, we attempted to develop a gene transfer system for HF epithelial stem cells to maximize the beneficial therapeutic effects. For persistent and stable transgene expression in HF stem cells, we transferred retroviral vectors encoding reporter genes into cultured HF stem cells. In addition, these cells were mixed with cultured dermal papilla cells and transplanted on to immunodeficient mice. We succeeded in reconstituting HFs and their appendages in which these cells harbored a transgene reporter. The transgene expression was observed in all skin epithelial compartments including the HF epithelium, sebaceous gland and epidermis. In addition, transgene expression was observed for at least 6 months. This HF stem cell-targeted gene transfer and reconstitution system provides reliable gene-function analysis and gene therapy.