Regulation of protocadherin gene expression by multiple neuron-restrictive silencer elements scattered in the gene cluster

The clustered protocadherins are a subfamily of neuronal cell adhesion molecules that play an important role in development of the nervous systems in vertebrates. The clustered protocadherin genes exhibit complex expression patterns in the central nervous system. In this study, we have investigated the molecular mechanism underlying neuronal expression of protocadherin genes using the protocadherin gene cluster in fugu as a model. By in silico prediction, we identified multiple neuron-restrictive silencer elements (NRSEs) scattered in the fugu protocadherin cluster and demonstrated that these elements bind specifically to NRSF/REST in vitro and in vivo. By using a transgenic Xenopus approach, we show that these NRSEs regulate neuronal specificity of protocadherin promoters by suppressing their activity in non-neuronal tissues. We provide evidence that protocadherin genes that do not contain an NRSE in their 5′ intergenic region are regulated by NRSEs in the regulatory region of their neighboring genes. We also show that protocadherin clusters in other vertebrates such as elephant shark, zebrafish, coelacanth, lizard, mouse and human, contain different sets of multiple NRSEs. Taken together, our data suggest that the neuronal specificity of protocadherin cluster genes in vertebrates is regulated by the NRSE-NRSF/REST system.     

Molecular imaging of gene expression and efficacy following adenoviral-mediated brain tumor gene therapy

Cancer gene therapy is an active area of research relying upon the transfer and subsequent expression of a therapeutic transgene into tumor cells in order to provide for therapeutic selectivity. Noninvasive assessment of therapeutic response and correlation of the location, magnitude, and duration of transgene expression in vivo would be particularly useful in the development of cancer gene therapy protocols by facilitating optimization of gene transfer protocols, vector development, and prodrug dosing schedules. In this study, we developed an adenoviral vector containing both the therapeutic transgene yeast cytosine deaminase (yCD) along with an optical reporter gene (luciferase). Following intratumoral injection of the vector into orthotopic 9 L gliomas, anatomical and diffusion-weighted MR images were obtained over time in order to provide for quantitative assessment of overall therapeutic efficacy and spatial heterogeneity of cell kill, respectively. In addition, bioluminescence images were acquired to assess the duration and magnitude of gene expression. MR images revealed significant reduction in tumor growth rates associated with yCD/5-fluorocytosine (5FC) gene therapy. Significant increases in mean tumor diffusion values were also observed during treatment with 5FC. Moreover, spatial heterogeneity in tumor diffusion changes were also observed revealing that diffusion magnetic resonance imaging could detect regional therapeutic effects due to the nonuniform delivery and/or expression of the therapeutic yCD transgene within the tumor mass. In addition, in vivo bioluminescence imaging detected luciferase gene expression, which was found to decrease over time during administration of the prodrug providing a noninvasive surrogate marker for monitoring gene expression. These results demonstrate the efficacy of the yCD/5FC strategy for the treatment of brain tumors and reveal the feasibility of using multimodality molecular and functional imaging for assessment of gene expression and therapeutic efficacy.     

Long-term tracing of adenoviral expression in rat and rabbit using luciferase imaging

BACKGROUND: Luciferase optical imaging provides a novel method to monitor transgene expression in small living animals. As the genetic and immunological heritages of particular animals significantly affect the expression of adenovirus-delivered transgenes, it is essential to know the expression patterns specific to athymic nude and Sprague-Dawley rats, two strains commonly used in rodent models. In this study we set out to determine these patterns. At the same time, we tested luciferase optical imaging in a larger animal, the rabbit. METHODS: A recombinant luciferase adenoviral vector was injected subcutaneously or intramuscularly into athymic nude rats, Sprague-Dawley rats, and Dutch Belted rabbits. The luciferase expression was assessed using a cooled charge-coupled device. RESULTS: The luminescent signal was capable of passing through at least 1.3 cm of muscle tissue and proved to be much stronger when luciferin was delivered via a local injection than by an intraperitoneal injection. Although the types of immune cells differed between immunodeficient and immunocompetent rats, similar amounts and patterns of luciferase expression were observed in the musculature in two rat strains during the 1st month after a viral intramuscular injection. The duration of luciferase expression was longer than 15 months in athymic nude rats, 9 months in Sprague-Dawley rats, and 6 months in rabbits following a direct viral injection. CONCLUSIONS: Luciferase expression after adenoviral gene delivery can persist for longer than 6 months, even in immunocompetent animals. Live imaging of luciferase expression can be performed not only in small animals, but also in larger animals such as rabbits.     

An Adenoviral Vector-Based System to Study Neuronal Gene Expression: Analysis of the Rat Tyrosine Hydroxylase Promoter in Cultured Neurons

Abstract: We validated an adenoviral vector-based system as a move toward the characterization of regulatory sequences that are involved in the control of cell-type specificity and ligand regulation of neuronal gene expression in cultured neurons. We constructed recombinant adenoviruses, incorporating the luciferase gene under the control of different fragments of the rat tyrosine hydroxylase (TH) promoter. Similar results for luciferase expression were obtained in immortalized cells either by infection using adenoviral constructs or by transfection using conventional plasmid vectors. Taking advantage of adenoviral vectors, we extended our experiments to various primary cell cultures. The first 800 bp of the TH promoter were found to be sufficient to confer a cell-type preferential activity in noradrenergic neurons of the rat superior cervical ganglia. Furthermore, using this neuronal culture model, we showed that the same promoter region carries leukemia-inhibitory factor (LIF)-responsive element(s). Our results demonstrate that the first 800 bp of the rat TH promoter contains a functionally important core region for constitutive and LIF-regulated expression of TH in peripheral noradrenergic neurons. Moreover, the study validates the adenoviral vector-based system as a new strategy for studying the regulation of neuronal gene expression.     

Saturation of transgene protein synthesis from mRNA in cells producing a large number of transgene mRNA

Experimental results have suggested that transgene expression can be saturated when large amounts of plasmid vectors are delivered into cells. To investigate this saturation kinetic behavior, cells were transfected with monitoring and competing plasmids using cationic liposomes. Even although an identical amount of a monitoring plasmid expressing firefly luciferase (FL) was used for transfection, transgene expression from the plasmid was greatly affected by the level of transgene expression from competing plasmids expressing renilla luciferase (RL). Similar results were obtained by exchanging the monitoring and competing plasmids. The competing plasmid‐dependent reduction in transgene expression from the monitoring plasmid was also observed in mouse liver after hydrodynamic injection of plasmids. On the other hand, the mRNA and protein expression level of glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH), an endogenous gene, in the liver hardly changed even when transgene expression process is saturated. The expression of FL from a monitoring plasmid was significantly restored by siRNA‐mediated degradation of RL mRNA that was expressed from a competing plasmid. These results suggest that the efficiency of protein synthesis from plasmid vectors is reduced when a large amount of mRNA is transcribed with no significant changes in endogenous gene expression. Biotechnol. Bioeng. 2011;108: 2380–2389. © 2011 Wiley Periodicals, Inc.     

Analysis of transcriptional regulatory sequences of the N-methyl-D-aspartate receptor 2A subunit gene in cultured cortical neurons and transgenic mice

The postnatal appearance and up-regulation of the NR2A subunit of the N-methyl-d-aspartate receptor contributes to the functional heterogeneity of the receptor during development. To elucidate the molecular mechanisms that regulate the neural and developmental specific expression of NR2A, an upstream approximately 9-kb region of the gene harboring the promoter was isolated and characterized in transgenic mice and transfected cortical neurons. Transgenic mouse lines generated with luciferase reporter constructs driven by either 9 or 1 kb of upstream sequence selectively transcribe the transgene in brain, as compared with other non-neural tissues. Reporter luciferase levels in dissociated cultures made from these mice are over 100-fold greater in neuronal/glial co-cultures than in pure glial cultures. Analysis of NR2A 5'-nested deletions in transfected cultures of cortical neurons and glia indicate that while sequences residing upstream of -1079 bp augment NR2A neuronal expression, sequences between -486 and -447 bp are sufficient to maintain neuronal preference. An RE1/NRSE element is not necessary for NR2A neuron specificity. Furthermore, comparison of the 5'-deletion constructs in cortical neurons grown for 5, 8, 11, or 14 days in vitro indicate that sequences between -1253 and -1180 bp are necessary for maturational up-regulation of NR2A. Thus, different cis-acting sequences control the regional and temporal expression of NR2A, implicating distinct regulatory pathways.     

Ocular Localization and Transduction by Adenoviral Vectors Are Serotype-Dependent and Can Be Modified by Inclusion of RGD Fiber Modifications

Purpose

To evaluate localization and transgene expression from adenoviral vector of serotypes 5, 35, and 28, ± an RGD motif in the fiber following intravitreal or subretinal administration.

Methods

Ocular transduction by adenoviral vector serotypes ± RGD was studied in the eyes of mice receiving an intravitreous or subretinal injection. Each serotype expressed a CMV-GFP expression cassette and histological sections of eyes were examined. Transgene expression levels were examined using luciferase (Luc) regulated by the CMV promoter.

Results

GFP localization studies revealed that serotypes 5 and 28 given intravitreously transduced corneal endothelial, trabecular, and iris cells. Intravitreous delivery of the unmodified Ad35 serotype transduced only trabecular meshwork cells, but, the modification of the RGD motif into the fiber of the Ad35 viral vector base expanded transduction to corneal endothelial and iris cells. Incorporation of the RGD motif into the fiber knob with deletion of RGD from the penton base did not affect the transduction ability of the Ad5 vector base. Subretinal studies showed that RGD in the Ad5 knob shifted transduction from RPE cells to photoreceptor cells. Using a CMV-Luc expression cassette, intravitreous delivery of all the tested vectors, such as Ad5-, Ad35- and Ad28- resulted in an initial rapid induction of luciferase activity that thereafter declined. Subretinal administration of vectors showed a marked difference in transgene activity. Ad35-Luc gene expression peaked at 7 days and remained elevated for 6 months. Ad28-Luc expression was high after 1 day and remained sustained for one month.

Conclusions

Different adenoviral vector serotypes ± modifications transduce different cells within the eye. Transgene expression can be brief or extended and is serotype and delivery route dependent. Thus, adenoviral vectors provide a versatile platform for the delivery of therapeutic agents for ocular diseases.     

Efficient adenoviral-mediated gene delivery into porcine mesenchymal stem cells

Mesenchymal stem cell (MSC) mediated gene therapy research has been conducted predominantly on rodents. Appropriate large animal models may provide additional safety and efficacy information prior to human clinical trials. The objectives of this study were: (a) to optimize adenoviral transduction efficiency of porcine bone marrow MSCs using a commercial polyamine-based transfection reagent (GeneJammer, Stratagene, La Jolla, CA), and (b) to determine whether transduced MSCs retain the ability to differentiate into mesodermal lineages. Porcine MSCs (pMSCs) were infected under varying conditions, with replication-defective adenoviral vectors carrying the GFP gene and GFP expression analyzed. Transduced cells were induced to differentiate in vitro into adipogenic, chondrogenic, and osteogenic lineages. We observed a 5.5-fold increase in the percentage of GFP-expressing pMSCs when adenovirus type 5 carrying the adenovirus type 35 fiber (Ad5F35eGFP) was used in conjunction with GeneJammer. Transduction of pMSCs at 10.3-13.8 MOI (1,500-2,000 vp/cell) in the presence of Gene Jammer yielded the highest percentage of GFP-expressing cells ( approximately 90%) without affecting cell viability. A similar positive effect was detected when pMSCs were infected with an Ad5eGFP vector. Presence of fetal bovine serum (FBS) during adenoviral transduction enhanced vector-encoded transgene expression in both GeneJammer-treated and control groups. pMSCs transduced with adenovirus vector in the presence of GeneJammer underwent lipogenic, chondrogenic, and osteogenic differentiation. Addition of GeneJammer during adenoviral infection of pMSCs can revert the poor transduction efficiency of pMSCs while retaining their pluripotent differentiation capacity. GeneJammer-enhanced transduction will facilitate the use of adenoviral vectors in MSC-mediated gene therapy models and therapies.