An Adenovirus Vector for Efficient RNA Interference–Mediated Suppression of Target Genes in Insulinoma Cells and Pancreatic Islets of Langerhans

RESEARCH DESIGN AND METHODS

Viral construction.

Relative to the start codon, the 5′ ends of the targets correspond to rat GLUT2 (accession number J03145) nucleotide 15 (GAT CAC CGG AAC CTT GGC T) and rat islet glucokinase (GK) (accession number M25807) nucleotide 202 (GGC TCA GAA GTC GGA GAC T). A target in the Photinus pyralis luciferase gene, GL2 (8), was used as a negative control. For each target, sense and antisense oligonucleotides were designed and synthesized, as described (9). Oligonucleotides were annealed in salt/Tris/EDTA buffer (10 mmol/l Tris, 1 mmol/l EDTA, 50 mmol/l NaCl, pH 8.0) and ligated into BglII/HindIII-linearized pSUPER (9; OligoEngine, Seattle, WA).

An adenoviral shuttle vector, EH006, was prepared by replacing the 1.2-kbp cytomegalovirus (CMV) promoter–based NotI expression cassette from pAC.CMV (10,15) with a mini-cloning cassette, constructed by annealing oligonucleotides TB468 (GGC CAT GGA TCC GAA TTC GTT TAA ACG AAG CTT TA) and TB469 (GGC CTA AAG CTT CGT TTA AAC GAA TTC GGA TCC AT) in STE.

For each pSUPER-based clone, the siRNA expression cassette was excised with EcoRI-HindIII and ligated into EcoRI-HindIII–linearized EH006 (Fig. 1A). To create RNAi adenoviruses by homologous recombination, EH006-derived shuttle vectors were cotransfected with the adenoviral plasmid, pJM17 (10), into human embryonic kidney cells (HEK293; American Type Culture Collection, Manassas, VA). Infectious titers of tertiary viral lysates, determined by end point dilution assay in HEK293 cells (16), ranged from 2.0 × 10⁹ to 1.0 × 10¹⁰ plaque-forming units (pfu)/ml.

Gene silencing in transgenic insulinoma cells.

The rat insulinoma line 832/13 was cultured as described (17). Cells in late log growth were plated at 52,600 cells/cm². One day later, the medium was removed and cells were transduced at viral doses of up to 240 pfu/cell for 2 h. Virus was removed, and fresh medium was added. At 24-h intervals, cells were lysed, RNA was isolated, and GLUT2 and GK transcript levels were assayed by real-time PCR analysis, using prevalidated primer and probes (Applied Biosystems). Three days after viral treatment, GK and GLUT2 protein levels were also evaluated by immunoblot analysis using polyclonal anti-GLUT2 (46701-659; Biogenesis, Poole, U.K.) and GK (GCK; Santa Cruz Biotechnology, Santa Cruz, CA) antisera and a horseradish peroxidase–conjugated, anti-rabbit secondary antibody (Amersham Biosciences, Little Chalfont, U.K.). Sample loading was monitored by measurement of total protein (Bio-Rad) and staining of gels with Coomassie Blue after protein transfer (not shown).

Gene silencing in rat islets of Langerhans.

Under a protocol approved by the Duke University Institutional Animal Care and Use Committee, pancreatic islets of Langerhans were isolated from male Wistar rats, 200–250 g, as described (18). Culture medium was that of Hohmeier et al. (17), with glucose adjusted to 7.2 mmol/l. Islets in aliquots of 360 per well of a six-well plate in 2 ml medium were transduced with recombinant RNAi adenoviruses for 20 h at a dose of 1.9 × 10⁶ pfu/islet. After transduction, islets were cultured for 3 days before measurement of GLUT2 protein levels by immunoblot, as described above.

RESULTS

Construction of siRNA-expressing adenoviruses.

We constructed an adenoviral shuttle vector, EH006 (Fig. 1A), that is designed to accept siRNA expression cassettes from the siRNA expression vector pSUPER (9). Importantly, EH006 was derived from the widely used adenoviral shuttle vector pAC.CMV (10,15) by replacement of the CMV-based expression cassette with a mini-cloning cassette, making this vector accessible via long-established and widely used methods and reagents. Recombinant viruses are created by cotransfection of HEK293 cells with an EH006-derived shuttle vector and pJM17 (10,15), a replication-defective variant of the adenoviral genome. After in vivo recombination, a recombinant viral genome is generated as shown in Fig. 1B. These viruses are predicted to express a short RNA hairpin (Fig. 1C), which is further processed to the bioactive siRNA by removal of the loop sequence by the mammalian homolog of the RNase III nuclease, Dicer.

Adenoviral-mediated silencing in insulinoma cells.

To test the ability of recombinant adenoviruses to direct RNAi-mediated gene silencing, we assayed GLUT2 and GK expression in an INS-1 insulinoma cell line, 832/13, after transduction with the Ad-GLUT2-siRNA and Ad-GK-siRNA viruses, respectively.

Treatment of 832/13 cells with the Ad-GLUT2-siRNA and Ad-GK-siRNA viruses caused decreases in GLUT2 and GK transcript levels in as little as 24 h after transduction, with maximal suppression of 70–80% observed 3 days after transduction (Fig. 2A). Compared with levels in untreated cells, we noted that GLUT2 and GK levels in Ad-luc-siRNA–transduced cells were slightly reduced 2 days after transduction, but, in both cases, GLUT2 and GK levels recovered by 3 days after transduction.

Treatment of 832/13 cells with Ad-GLUT2-siRNA reduced GLUT2 protein levels by 80% (Fig. 2B), whereas treatment with Ad-GK-siRNA reduced GK protein levels by 50% (Fig. 2C). Treatment of the same cells with a control virus (Ad-luc-siRNA) had no effect on GLUT2 or GK protein levels. Because GLUT2 and GK RNA levels were reduced to a similar extent after transduction, the lesser efficiency of suppression of GK protein levels is likely attributable to differences in rates of protein turnover.

Adenoviral-mediated RNAi silenced GLUT2 expression in cultured rat islets.

Although important insights can be gained by genetic engineering studies in insulinoma cell lines, key concepts must always be confirmed in primary cell preparations. We therefore tested the utility of our new adenovirus vectors for delivery of siRNAs into cultured rat islets. As shown in Fig. 3, treatment of cultured islets with 1.9 × 10⁶ pfu/islet of Ad-GLUT2-siRNA resulted in a >95% reduction in GLUT2 expression compared with untreated islets and a >90% reduction when compared with Ad-luc-siRNA–treated islets.

DISCUSSION

Pancreatic islets of Langerhans play a critical role in control of metabolic fuel homeostasis. Islet function and mass are lost or disturbed in diabetes, and this has stimulated research efforts focused on identification of genes that can enhance islet function and survival. Multiple reports have appeared on microarray analysis for identification of genes involved in regulation of islet cell development (1), insulin secretion (2,3), and stress responses (4,5) in β-cell model systems. However, functional studies that prove the involvement of specific genes in defining the phenotype under study have been lacking. This report describes a vector system that should be useful for defining the “functional genomics” of the β-cell, since it allows rapid analysis of candidate genes in tractable in vitro systems, including primary preparations of pancreatic islets. Although a number of researchers have previously described adenoviral-based RNAi systems (19,20), the current study describes an adenoviral RNAi shuttle vector that is based on a well-established adenoviral platform (10,15) already in widespread use among diabetes researchers (21–24) and is the first to describe siRNA-mediated gene suppression in primary islet cells. Moreover, the suppression of one target gene (GLUT2) by more than 90% in primary cultures of rat islets strongly supports the utility of our methods for gene silencing within β-cells, since these cells constitute ∼70–80% of the total islet cell population and are the main site of islet GLUT2 expression.

A number of other RNAi viral systems have also been previously described, including retroviral (25) and lentiviral (26,27) systems. The genomes of these viruses integrate into the host genome, leading to long-term expression, in contrast to the adenoviral genome, which remains episomal, resulting in relatively transient expression. However, recombinant adenoviruses can be grown to much higher titers than lenti- and retroviruses, and this characteristic may make adenoviruses particularly useful tools for RNAi-mediated gene silencing, since intracellular viral copy number and therefore expression of siRNAs may be significantly higher. Adenoviral reagents that drive overexpression of selected genes have proven themselves useful in metabolic research into diabetes and obesity, both in vitro and in vivo (10–14,28). Data in this report demonstrate that islets and insulinoma cell lines can be targeted for selective gene suppression using recombinant RNAi adenoviruses.