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PEG-g-PLL Complex
doi:10.1006/mthe.2001.0458, available online at http://www.idealibrary.com on IDEAL
ARTICLE
Repression of GAD Autoantigen Expression in Pancreas -Cells by Delivery of Antisense Plasmid/PEG-g-PLL Complex Minhyung Lee,1 Sang-oh Han,1 Kyung Soo Ko,1 Jae Joon Koh,1 Jong-Sang Park,2 Ji-Won Yoon,3 and Sung Wan Kim1,* 1
Center for Controlled Chemical Delivery, Department of Pharmaceutics and Pharmaceutical Chemistry, University of Utah, Salt Lake City, Utah 84112, USA 2 Department of Chemistry, Seoul National University, Seoul 151-742, Korea 3 Julia-Mcfarlane Diabetes Research Center, University of Calgary, Calgary, Alberta, Canada T2N 4N1 *To whom correspondence and reprint requests should be addressed. Fax: (801) 581-7848. E-mail: [email protected].
It was previously reported that silencing of the expression of glutamic acid decarboxylase (GAD) in transgenic nonobese diabetic (NOD) mice completely protected islet -cells against development of diabetes. This suggests that the repression of GAD autoantigen by somatic gene delivery can prevent autoimmune destruction of pancreatic -cells. To repress GAD expression in islet -cells, we delivered an antisense GAD mRNA expression plasmid (pRIP-AS-GAD) using poly(ethylene glycol)-grafted poly-L-lysine (PEG-g-PLL) as a gene carrier. In a gel retardation assay, the pRIP-AS-GAD/PEG-g-PLL complex was completely retarded above a weight ratio of 1:1.5 (plasmid:PEG-g-PLL). PEG-g-PLL protected the plasmid DNA from DNase I for more than 60 minutes. In a reporter gene transfection assay, PEG-g-PLL showed the highest transfection efficiency at a weight ratio of 1:3. We also transfected pRIP-AS-GAD/PEG-g-PLL complex into a GAD-producing mouse insulinoma (MIN6) cell line. The antisense mRNA was expressed specifically in -cells and expression was dependent on glucose level. The repression of GAD after transfection of pRIPAS-GAD was confirmed by immunoblot assay. In addition, in vivo expression of antisense RNA in pancreas was confirmed by RT-PCR after intravenous injection of the complex into mice. Therefore, our study revealed that the pRIP-AS-GAD/PEG-g-PLL system is applicable for the repression of GAD autoantigen expression. Key words: antisense plasmid, autoimmune diabetes, glutamic acid decarboxylase (GAD), gene delivery, pancreatic -cell, polyethylene glycol-grafted poly-L-lysine (PEG-g-PLL)
INTRODUCTION Type 1 (insulin-dependent) diabetes mellitus results from autoimmune destruction of the -cells of the islets of Langerhans. The destructive process is thought to be immune mediated [1]. Glutamic acid decarboxylase (GAD) 65 has been identified as a major autoantigen in type I diabetes, where GAD65-specific antibodies are present in about 80% of newly diagnosed patients [2–4]. GAD catalyzes the decarboxylation of L-glutamic acid to ␥aminobutyric acid (GABA), the major inhibitory neurotransmitter in the mammalian brain. Two isoforms, named GAD65 and GAD67, have been cloned and the human isoforms are approximately 65% identical at the amino acid level [5,6]. They can be distinguished by their molecular masses, their cofactor interactions, and their subcellular distributions [7,8]. GAD65 is largely membranebound and is relatively enriched in vesicular membranes,
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whereas GAD67 is soluble and distributed in the cytoplasm. GAD is expressed primarily in GABAnergic neurons of the adult mammalian central nervous system, but is also present in pancreatic -cells and testes [9,10]. It was reported that anti-GAD monoclonal antibody delays the onset of diabetes mellitus in nonobese diabetic (NOD) mice [11]. In addition, silencing of the expression of GAD65 and GAD67 in the -cells completely protected against development of diabetes in the transgenic NOD mouse, suggesting that GAD has a major role in the development of type 1 diabetes mellitus (IDDM) [12]. These data suggest that the repression of GAD autoantigen by somatic gene delivery of antisense expression plasmid can prevent autoimmune destruction of pancreatic -cells. Delivery of antisense DNA has been widely used to control specific gene expression [13]. The principle of antisense inhibition relies on the specificity of base-pair formation
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FIG. 1. Diagram of pRIP-AS-GAD structure. RIP, rat insulin promoter; I/A, polyadenylation signal.
[14–16]. This specificity enables antisense nucleic acids to inhibit the transcription or translation of specific genes. Synthetic oligonucleotides or antisense expression plasmid has been used for antisense inhibition, but the major limitation of antisense inhibition is that it is difficult to deliver sufficient amounts of antisense DNA for substantial inhibition of the expression of a specific gene. This is due to the inefficiency of naked DNA entry into the cells. Therefore, most antisense studies have used modified DNA structures to increase the availability and the stability of the nucleic acid [17–19]. Phosphorothioate is one of the most frequent variants of antisense oligonucleotides [16,18,20–22]. Cationic lipid or polymer was also used to improve the stability and availability of the nucleic acid [17,23]. Poly(ethylene glycol)-grafted poly-L-lysine (PEG-g-PLL) is a polymeric gene carrier [24] that has lower cytotoxicity and higher transfection efficiency than PLL. Here we synthesize three kinds of PEG-g-PLLs with various PEG contents. We evaluated complex formation and stabilization effects of these PEG-g-PLLs. Antisense expression plasmid/PEG-g-PLL complex was delivered to GAD-producing cells to repress GAD expression. The antisense GAD mRNA expression was driven by the rat insulin gene promoter for cell-specific and glucose-concentration-dependent expression. The transfection conditions were optimized for the delivery of antisense expression plasmid. The expression of antisense GAD mRNA was confirmed by RT-PCR, and GAD protein level is measured by immunoblot assay. In addition, in vivo expression of antisense GAD mRNA in pancreas was confirmed by RT-PCR after the intravenous injection of the complex into mice.
We carried out a gel retardation assay to investigate whether the synthesized PEG-g-PLL forms a complex with the antisense expression plasmid, pRIP-AS-GAD. pRIP-ASGAD consists of GAD65 and GAD67 cDNAs in the antisense direction, the rat insulin gene promoter, and polyadenylation signals (Fig. 1). The band of plasmid DNA was retarded as the amount of PEG-g-PLL increased, suggesting that PEG-g-PLL forms a complex with plasmid DNA (Fig. 2). The plasmid band was completely retarded above a 1:1.5 weight ratio of plasmid DNA:PEG-g-PLL (Fig. 2, lanes 4–7). A DNase I protection assay was carried out to confirm that PEG-g-PLL protected plasmid DNA from DNase I. The protection efficiencies of 10, 15, and 20 mole percent (mol%) PEG-g-PLL were evaluated. After complex formation at a 1:1.5 weight ratio (plasmid DNA:PEG-g-PLL), the plasmid DNA/PEG-g-PLL complexes were incubated with DNase I for 20 or 60 minutes. Naked DNA was completely degraded by DNase I after 20 minutes of incubation (Fig. 3, lanes 2 and 3), but 10 mol% PEG-g-PLL protected plasmid DNA over 60 minutes (Fig. 3, lanes 4 and 5). We found that 15 and 20 mol% PEG-g-PLLs were less effective than 10 mol% PEG-g-PLL in protection (Fig. 3, lanes 6–9). Transfection Assay with pSV--galactosidase/PEG-gPLL Complex To determine the most effective weight ratio of plasmid DNA:polymer, a reporter gene transfection assay was carried out. We used pSV--galactosidase as a reporter gene for monitoring gene expression in 293T cells. In pSV-galactosidase, the expression of lacZ (encoding -galactosidase) was driven by SV40 early promoter and enhancer. Various plasmid DNA/polymer complexes were formulated with a fixed amount of plasmid DNA (20 g) and increasing amounts of 10 mol% PEG-g-PLL (20–120 g). A 1:3
RESULTS Synthesis and Characterization of PEG-g-PLL We synthesized three kinds of PEG-g-PLLs with various PEG contents and characterized them by 1H-NMR. The content of PEG was calculated from the 1H-NMR spectrum.
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FIG. 2. Gel retardation assay. pRIP-AS-GAD plasmid (1 g) was mixed with various quantities of PEG-g-PLL. After incubation at room temperature for 30 min, the complexes were analyzed by 0.7% agarose gel electrophoresis. Lane 1, plasmid-only control.
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FIG. 3. DNase I protection assay. pRIP-AS-GAD/PEG-g-PLL complex solution was incubated with DNase I for 20 min or 60 min. After incubation, DNA was analyzed by 0.7% agarose gel electrophoresis.
Expression of GAD in MIN6 cells was also confirmed by an immunoblot assay. Cell extracts from MIN6 cells or BALB/c mouse islets were separated in 10% polyacrylamide gel and analyzed with anti-GAD antibody. Although the expression level of GAD in MIN6 cell extract was lower than that in mouse islet extract, GAD65 and GAD67 were detected in both extracts (Fig. 6B, lane 1 and 2). Therefore, MIN6 cells can be used as an in vitro GAD-producing cell model.
weight ratio of plasmid DNA:PEG-g-PLL showed the most effective transfection (Fig. 4). This result was exactly the same as in a previous report [24]. In this previous report, in which 5, 10, and 25 mol% PEG-g-PLLs were used, three kinds of PEG-g-PLLs showed little difference in complex formation ability above a 1:3 weight ratio (plasmid DNA:PEG-g-PLL) [24]. In addition, 10 mol% PEG-g-PLL showed the highest transfection efficiency at a 1:3 weight ratio (plasmid DNA:PEG-g-PLL). Therefore, we carried out the transfection assay with 10, 15, and 20 mol% PEG-g-PLL at a 1:3 weight ratio (plasmid DNA:PEG-g-PLL) to determine which PEG-g-PLL was the most effective in transfection. The complexes were transfected into MIN6 cells. Among those tested, the 10 and 15 mol% PEG-g-PLL showed almost the same transfection efficiency (Fig. 5), but 20 mol% PEG-g-PLL was less effective than 10 and 15 mol% PEG-g-PLL. Therefore, we used 10 mol% PEG-g-PLL in the following experiments, because it protected plasmid DNA from DNase I most effectively and showed high transfection efficiency.
Antisense GAD mRNA Expression in MIN6 Cells We transfected pRIP-AS-GAD/PEG-g-PLL complex into MIN6 cells and carried out RT-PCR to detect the antisense GAD67 mRNA. We mixed 5, 10, or 20 g of pRIP-AS-GAD plasmid with PEG-g-PLL at a weight ratio of 1:3 and transfected into MIN6 cells. At 48 hours after transfection, the cells were collected and total RNA was prepared. To determine the expression level, antisense GAD67 mRNA was amplified. We identified a 357-bp PCR product in agarose gel electrophoresis. The level of antisense GAD67 mRNA increased with increasing amounts of plasmid (Fig. 7A). Duration of antisense GAD mRNA expression was also evaluated by RT-PCR (Fig. 7B). Although the expression level of antisense GAD mRNA decreased, antisense GAD mRNA was detected in the cells at 96 hours after transfection. Antisense mRNA expression was driven by the rat insulin gene promoter. Therefore, the antisense GAD mRNA expression was tissue (pancreas islet)-specific and responsive to glucose concentration. To confirm the tissuespecific expression, pRIP-AS-GAD/PEG-g-PLL complexes were transfected to MIN6 cells or 293T cells and the
GAD Expression in MIN6 Cells Before using MIN6 cells as an in vitro GAD-producing cell model, we confirmed by RT-PCR that MIN6 cells produce GAD antigen. Total RNA was prepared from MIN6 cells and used as a template. We identified 293-bp and 357bp PCR products, which are the expected sizes for GAD65 and GAD67 mRNAs, respectively. This result suggested that GAD65 and GAD67 were produced in MIN6 cells (Fig. 6A, lanes 1–4). -Actin mRNA was amplified as an endogeneous control (Fig. 6A, lanes 5 and 6).
FIG. 4. Effect of plasmid DNA:PEG-g-PLL weight ratio on transfection of 293T cells. DNA/PEG-g-PLL complexes were formulated with a fixed amount of plasmid DNA (20 g) and increasing amounts of 10 mol% PEG-g-PLL (20–120 g). After 30 min of incubation at room temperature, the complexes were transfected to 293T cells. Transfection efficiency was measured by -galactosidase assay. Bar, standard error of mean.
MOLECULAR THERAPY Vol. 4, No. 4, October 2001 Copyright © The American Society of Gene Therapy
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FIG. 5. Effect of PEG contents in PEG-g-PLL on transfection efficiency of MIN6 cells. DNA/PEG-g-PLL complexes were formulated with plasmid DNA (20 g) and polymer (60 g). After 30 min of incubation at room temperature, the complexes were transfected to MIN6 cells. Transfection efficiency was measured by -galactosidase assay. Bar, standard error of mean.
the transfected cells. We transfected 5, 10, or 20 g pRIPAS-GAD into MIN6 cells by formation of complexes with PEG-g-PLL. At 48 hours after transfection, the cell extracts were prepared and analyzed with anti-GAD antibody. The expression levels of GAD65 and GAD67 decreased significantly with increasing amounts of pRIP-AS-GAD (Fig. 8).
expressed antisense GAD mRNAs were amplified by RTPCR. We detected a PCR product of 357 bp for antisense GAD67 mRNA in RT-PCR with total RNA from MIN6 cells (Fig. 7C, lane 3). However, RT-PCR with total RNA from 293T cells did not show any signal (Fig. 7C, lane 2). To confirm the glucose concentration responsive expression of antisense GAD mRNA, the transfected cells were maintained in medium containing 0, 1, 5, or 25 mM glucose. The expression level of antisense GAD mRNA was measured by RT-PCR (Fig. 7D), revealing that the expression level increased as the glucose level increased in the medium. Therefore, antisense GAD mRNA is expressed in response to the glucose level in the medium.
In Vivo Expression of Antisense mRNA in Mouse Pancreas after Injection of pRIP-AS-GAD/PEG-g-PLL Complex To evaluate the expression of antisense GAD mRNA in mouse pancreas, pRIP-AS-GAD/PEG-g-PLL complex was injected intravenously into mice. PEG-g-PLL was injected into mice as a control. To detect antisense GAD mRNA, RTPCR was carried out with total RNA prepared from the pancreas. Antisense RNA was detected in the pancreas of mice injected with pRIP-AS-GAD/PEG-g-PLL complex after 1 or 3 days of injection (Fig. 9). However, in the pancreas of mice injected with PEG-g-PLL only, antisense RNA was not detected. These results suggest that the pRIP-ASGAD/PEG-g-PLL complex is applicable to in vivo repression of GAD antigen.
DISCUSSION
Repression of GAD in MIN6 Cells by Transfection of pRIP-AS-GAD/PEG-g-PLL Complex To evaluate the repression of GAD in MIN6 cells, an immunoblot assay was carried out with cell extracts from
Antisense repression of GAD has been tried by several researchers on the field of the nervous system [25–29]. These studies were designed to evaluate the effect of the changes of GABA and glutamate concentrations on the differentiation of the nervous system, food intake, or locomotive behavior. In all of these studies, antisense oligonucleotides were used. Also, it was reported that
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FIG. 6. Expression of GAD in MIN6 cells. (A) Detection of GAD65, GAD67, and -actin mRNAs in MIN6 cells by RT-PCR. Total RNA was prepared from MIN6 cells. Total RNA was reverse transcribed and amplified by PCR using specific primers. Lanes 1 and 2, GAD67; lanes 3 and 4, GAD65; lanes 5 and 6, -actin. M, DNA size marker. (B) Detection of GAD65 and GAD67 in mouse islet and MIN6 cells by immunoblot assay. Cell extracts (40 g) were separated in 10% SDS-polyacrylamide gel and transferred to PVDF membrane. GAD65 and GAD67 were detected by immunoblot assay.
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FIG. 7. Expression of antisense GAD mRNA in MIN6 cells after transfection. (A) Dose-dependent antisense GAD mRNA expression. Total RNA was prepared from transfected MIN6 cells. GAD67 mRNA was reverse transcribed and amplified by PCR using specific primers. M, DNA size marker. (B) Duration of antisense GAD mRNA expression. Total RNA was prepared from transfected MIN6 cells at the indicated time and analyzed by RT-PCR. M, DNA size marker. (C) Cell-type-specific antisense GAD mRNA expression. pRIP-AS-GAD/PEG-gPLL complexes were transfected to 293T cells (lane 2) or MIN6 cells (lane 3). Total RNA was prepared from 293T cells or MIN6 cells and analyzed by RTPCR. M, DNA size marker. (D) Glucose-responsive antisense GAD mRNA expression. MIN6 cells were cultured in glucose-containing medium as indicated. At 48 h after transfection, total RNA was prepared and analyzed by RTPCR. M, DNA size marker; lane 1, negative control without transfection.
silencing of the expression of GAD in transgenic NOD mice with an antisense GAD transgene completely protected islet -cells against the development of autoimmune diabetes [12]. However, somatic gene delivery of antisense expression plasmid for prevention of type 1 diabetes has not been reported. In our study, pRIP-AS-GAD plasmid instead of antisense oligonucleotides was delivered to GAD-producing MIN6 cells by complex formation with PEG-g-PLL. This pRIP-AS-GAD/PEG-g-PLL system has advantages over modified antisense oligonucleotides. Unlike the antisense oligonucleotide approach, this system allows delivery of a larger size of DNA using PEG-g-PLL as a gene carrier. The antisense oligonucleotide system uses 15–25 bp oligonucleotides. Therefore, the oligonucleotides should be designed to be complementary to the most effective site for inhibition of GAD expression. However, in the pRIP-AS-GAD/PEG-g-PLL system, whole antisense cDNA was delivered and, therefore, antisense mRNA can hybridize with the sense mRNA effectively. In addition, the
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delivery of larger sizes of DNA allowed the insertion of the rat insulin gene promoter into the plasmid. As a result, the antisense mRNA was expressed specifically in pancreas. Also, the insulin gene promoter has been reported to respond to the glucose level [30–32], and we confirmed that expression of antisense GAD mRNA was induced by increasing glucose concentration (Fig. 7D). We synthesized three kinds of PEG-g-PLL and characterized them to optimize the antisense plasmid transfection. The protective effect of PEG-g-PLL was evaluated by DNase I protection assay (Fig. 3). Previously, DNase I was demonstrated as one of many nucleases existing in serum [33]. Therefore, the stability of the plasmids in the presence of DNase I is a critical parameter for in vivo gene delivery. After DNase I treatment, 10 mol% PEG-g-PLL protected the plasmid DNA most effectively (Fig. 3); 15 or 20 mol% PEG-g-PLL was less effective in protection of DNA. It was expected that higher mol% PEG-g-PLLs would require a higher plasmid DNA:polymer ratio to achieve effective DNA protection.
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FIG. 8. Repression of GAD antigen expression. At 48 h after transfection, cell extracts were prepared from the transfected cells. Extracts were separated in 10% SDS-polyacrylamide gel and transferred to PVDF membrane. GAD67 and GAD65 were detected by immunoblot assay.
We found that 10 mol% PEG-g-PLL showed the highest transfection efficiency at a weight ratio of 1:3 (plasmid DNA:PEG-g-PLL) in a reporter gene transfection assay (Fig. 4). At this weight ratio, pRIP-AS-GAD/PEG-g-PLL complex was formed and transfected to MIN6 cells. It has not been reported that MIN6 cells produce GAD autoantigen. However, we confirmed the expression of GAD in MIN6 cells by RT-PCR and immunoblot assay (Figs. 6A and 6B). Therefore, MIN6 cells are a good in vitro GAD-producing cell model for evaluation of GAD repression. pRIP-ASGAD/PEG-g-PLL expressed antisense GAD mRNA over 96 hours in MIN6 cells and repressed the expression of GAD antigen at a substantial level. After the injection, the expression of antisense mRNA was evaluated by RT-PCR. The antisense mRNA was expressed in the pancreas of mice injected with pRIP-ASGAD/PEG-g-PLL complex. The antisense RNA was expressed for more than 3 days, suggesting that pRIP-ASGAD/PEG-g-PLL complex is applicable to in vivo repression of GAD antigen. A previous report showed that the pancreatic insulin content and plasma insulin concentrations of RIP-AS-GAD transgenic mice were indistinguishable from those of transgene negative NOD mice [12]. This suggests that repression of GAD expression does not have any side effects on normal function of pancreas. Also, GAD expression in brain was detected equally in brain tissue of transgene-negative NOD mice and RIP-AS-GAD transgenic NOD mice, suggesting that the repression of GAD by pRIP-ASGAD/PEG-g-PLL may not have any effect on normal expression of GAD in other organs [12]. Therefore, the pRIP-AS-GAD/PEG-g-PLL system is effective to repress the expression of GAD autoantigen specifically in pancreatic -cells.
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FIG. 9. Expression of antisense GAD mRNA in pancreas of mice after the intravenous injection of pRIP-AS-GAD/PEG-g-PLL complex. Total RNAs were prepared from the pancreas of the mice injected with pRIP-AS-GAD/PEG-g-PLL complex or PEG-g-PLL only. GAD67 mRNA was reverse transcribed and amplified by PCR using specific primers. M, DNA size marker.
MATERIALS
AND METHODS
Preparation of pRIP-AS-GAD. The construction of the antisense GAD expression plasmid has been described [12]. The antisense expression plasmid, pRIP-AS-GAD, consists of GAD65 and GAD67 cDNAs in the antisense direction, the rat insulin gene promoter, and polyadenylation signals (Fig. 1). The expression of antisense mRNA was driven by the rat insulin gene promoter. pRIP-AS-GAD was introduced into Escherichia coli strain JM109 and isolated in large quantities using the plasmid purification Maxi kit (Qiagen, Valencia, CA). Purity of plasmids was certified by OD260/OD280 ratio and by distinctive bands of DNA fragments at corresponding base pairs in agarose gel electrophoresis following XhoI and NotI digestion. The concentration of plasmid DNA was determined using OD260 = 50 g of DNA. The plasmid DNA was stored at –20⬚C until use. Synthesis of PEG-g-PLL. We prepared three different types of PEG-g-PLL with various PEG contents of 10, 15, and 20 mol% as described [24] with slight modification. Synthesis of 10 mol% PEG-g-PLL is described as an example. PLL-hydrobromide (100 mg) was dissolved in 1 ml DMSO and 10 l TEA and stirred for 30 min. M-SPA-2000 (100 mg) was dissolved in 2 ml DMSO and added into PLL solution for 30 min. The mixed solution was stirred at room temperature for 6 h. The resulting material was precipitated in excess of ethylether. The material was washed with ether three times and then with methylene chloride three times. The precipitated polymer was dried and dissolved in 2 ml deionized water. The synthesized PEG-g-PLL was purified by dialysis against deionized water, followed by lyophilization. The polymer was characterized the contents of PEG by 1H-NMR in D2O. Gel retardation assay. Increasing amounts of PEG-g-PLL, ranging from 0.5 to 3 g, were added to 1 g pRIP-AS-GAD plasmid DNA. The mixtures were incubated at room temperature for 30 min and electrophoresed on 0.7% (w/v) agarose gel. DNase I protection assay. The weight ratio of plasmid/PEG-g-PLL complex was 1:3 (plasmid:PEG-g-PLL) in phosphate buffered saline (PBS). After complex formation, DNase I (10 units, Gibco BRL, Gaithersburg, MD) was added to the complex solution and the reaction mixture was incubated at 37⬚C. The sample (50 l) was taken at 20 or 60 min after incubation, mixed with 50 l of 2⫻ stop solution (0.4 M NaCl, 80 mM EDTA, and 2% SDS), and placed on ice. To dissociate the plasmid DNA from PEG-g-PLL, the mixtures were incubated at 60⬚C overnight. After phenol/chloroform extraction, the DNA was precipitated with ethanol. The pellets were dissolved in 10 l TrisEDTA (TE) buffer and applied to the 0.7% agarose gel electrophoresis.
MOLECULAR THERAPY Vol. 4, No. 4, October 2001 Copyright © The American Society of Gene Therapy
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Cell culture. MIN6 cells and 293T cells were maintained in DMEM supplemented with 15% (MIN6 cells) or 10% (293T cells) FBS in a 5% CO2 incubator. MIN6 cells retain glucose-inducible insulin secretion like normal cells [34]. For the transfection studies, MIN6 or 293T cells were seeded at a density of 2.0 ⫻ 106 cells/100-mm culture dish and incubated for 24 h before the addition of the plasmid DNA/polymer complex. Transfection. The in vitro transfection assay was carried out as follows. Plasmid DNA/PEG-g-PLL complexes were prepared by mixing 20 g of plasmid DNA and various amounts of PEG-g-PLL (20, 40, 60, or 120 g) in 500 l of serum-free DMEM medium, and incubated for 30 min at room temperature. The MIN6 or 293T cells were washed twice with serum-free DMEM medium, then 10 ml fresh serum-free medium was added. Plasmid DNA/PEG-g-PLL (500 l) complex was added to each dish. The cells were then incubated for 4 h at 37⬚C in a 5% CO2 incubator. After 4 h, the transfection mixtures were removed and 10 ml of fresh DMEM medium containing FBS was added to each dish. Cells were incubated for a desired time at 37⬚C. -Galactosidase assay. Cells in each dish were washed twice with PBS and 900 l of 1⫻ reporter lysis buffer (Promega, Madison, WI) was added to cover the cells. After 15 min of incubation at room temperature, the cells were collected and transferred to a microcentrifuge tube. After 15 s of vortexing, the cells were centrifuged at 12,000g for 2 min. The extracts were transferred to a fresh tube and stored at –70⬚C until use. The protein concentrations of the extracts were determined using the BCA protein assay kit (Pierce, Rockford, IL). -Galactosidase assay mixtures included 100 g cell extracts and 150 l of assay 2⫻ buffer (Promega, Madison, WI) in 300 l of volume. The mixtures were incubated at 37⬚C for 90 min. After the reaction, 500 l of 1 M sodium carbonate was added to the mixtures and the absorbance at 420 nm was measured. RT-PCR. The transfected cells were washed twice with PBS and total RNA was prepared by acid-guanidium thiocyanate-phenol-chloroform extraction as described [35], using RNAwiz (Ambion, Austin, TX). The total RNA was treated with RNase-free DNase I (Promega, Madison, WI) to eliminate the contaminated DNA. The concentration of RNA was measured by the absorbance at 260 nm, and the integrity of RNA was confirmed by formaldehyde-formamide denatured agarose gel electrophoresis. Total RNA (2 g) was hybridized to the backward primer and reverse transcribed using AMV reverse transcriptase (Promega, Madison, WI). The reverse-transcribed samples were amplified by PCR using Taq polymerase (Life Promega, Madison, WI). The sequences of the specific oligonucleotide primers were as follows: GAD67 forward primer, 5⬘-ATGACGTCTCCTACGATACA-3⬘; GAD67 backward primer, 5⬘-CCCCTTGAGGCTGGTAACCA-3⬘; GAD65 forward primer, 5⬘-AAGGGGACTACTGGATTTGA-3⬘; GAD65 backward primer, 5⬘-TGCGGAAGAAGTTGACCTTA-3⬘; -actin forward primer, 5⬘-TGGAATCCTGTGGCATCCATGAAA C-3⬘; -actin backward primer, 5⬘TAAAACGCAGCTCAGTAACAGTCCG-3⬘. The PCR reaction consisted of 94⬚C for 3 min, 35 cycles at 94⬚C for 30 s, 58⬚C for 30 s, and 72⬚C for 1 min, followed by an extension of 10 min at 72⬚C. The PCR products were separated by electrophoresis in 2% agarose gels. The lengths of the expected products were 357 bp for GAD67, 293 bp for GAD65, and 354 bp for -actin. Anti-GAD antibody production and purification. A hybridoma cell line producing anti-GAD antibody (HB184, IgG1, anti-GADF-6 that is GAD65 and GAD67 specific) was obtained from ATCC (Rockville, MD). The hybridoma cell line was injected into the peritoneum of Balb/c mice to produce anti-GAD antibody in the ascites fluid. Anti-GAD antibody was extracted and purified after precipitation with saturated ammonium sulfate, followed by anion exchange chromatography and protein A-Sepharose chromatography, as described [36]. The purified fractions were pooled, dialyzed, and filtered with 0.2-m sterile Acrodisk membranes, diluted to a concentration of 2 mg/ml in PBS, and stored at –20⬚C. The purified fractions were characterized by electrophoresis (Phastgel 8/25, Pharmacia, Piscataway, NJ) under native and denaturing conditions, and with a homogeneous gel in denaturing and reducing conditions. Islet isolation. Islets of Langerhans were isolated from male Balb/c mouse pancreas by a modified collagenase digestion technique and discontinuous Ficoll density gradient centrifugation [37]. Briefly, the pancreas was removed after swelling by collagenase solution injection (5–7 ml/pancreas,
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1 mg/ml) through the common bile duct and incubated for 15 min at 37⬚C. The digested fragments of the pancreas were collected and washed with HBSS. Finally, the acinar cells and islets were separated by Ficoll density gradient centrifugation. The islet-rich layer was collected and washed with HBSS, and islets were suspended in RPMI-1640 medium with 10% FBS and incubated at 37⬚C under humidified conditions with 5% CO2. On average 100–150 islets were isolated from a mouse pancreas. Immunoblot assays. The cells were washed with PBS twice and 1.0 ml lysis buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.02% sodium azide, 100 g/ml phenylmethanesulfonic fluoride (PMSF), 1 g/ml aprotinin, and 1% Nonidet P-40) was added to each dish. The cells were incubated for 20 min on ice. The cells were harvested and transferred to a new tube. For the preparation of cell extracts from pancreas, pancreas was homogenized in lysis buffer and transferred to a new tube. After the centrifugation at 12,000g for 2 min at 4⬚C, the extracts were transferred to a new tube. The extracts were stored at –70⬚C until use. The protein concentrations of the extracts were determined by using BCA protein assay kit (Pierce, Rockford, IL). Cell extracts (40 g) from transfected cells were subjected to electrophoresis in a 10% SDS-polyacrylamide gel. For western blot analysis, proteins were transferred to a polyvinylidene difluoride membrane (PVDF, Millipore, Bradford, MA) for 60 min at 300 mA in transfer buffer (10% methanol, 0.01 M 2-[cyclohexylamino]-1-propane-sulfonic acid (CAPS), pH 11). The membrane was washed with PBST (0.01% Tween 20 in PBS) and blocked overnight in blocking buffer (5% non-fat dried milk in PBS). The membrane was incubated in primary antibody solution for 90 min, and then in secondary antibody solution (anti-mouse IgG, Sigma, St. Louis, MO) for 30 min. The bands were visualized by amplified Opti-4CN detection kit (Bio-Rad, Hercules, CA) as described in the manufacturer’s manual. In vivo expression of GAD mRNA in pancreas of mice. pRIP-ASGAD/PEG-g-PLL complexes were prepared at a weight ratio of 1:3 (plasmid:PEG-g-PLL). pRIP-AS-GAD/PEG-g-PLL complexes (200 l) were injected into the tail vein of Balb/c mice at a dose of 50 g DNA per mouse. At designated times, the animals were killed by cervical dislocation and the pancreas was harvested. The expression of antisense GAD mRNA was evaluated by RT-PCR as described above.
ACKNOWLEDGMENTS We thank Jun-Ichi Miyazaki (Osaka University Medical School, Osaka, Japan) for MIN6 cells, Troy Koch (University of Utah) for technical assistance, and Expression Genetics, Inc., for financial support. This work was supported by National Institutes of Health grant DK51689. RECEIVED FOR PUBLICATION JANUARY 19; ACCEPTED JULY 23, 2001.
REFERENCES 1. Atkinson, M. A., and Maclaren, N. K. (1994). The pathogenesis of insulin-dependent diabetes mellitus. N. Engl. J. Med. 331: 1428–1436. 2. Verge, C. F., et al. (1994). Anti-glutamate decarboxylase and other antibodies at the onset of childhood IDDM: a population-based study. Diabetologia 37: 1113–1120. 3. Hagopian, W. A., et al. (1995). Glutamate decarboxylase-, insulin-, and islet cell-antibodies and HLA typing to detect diabetes in a general population-based study of Swedish children. J. Clin. Invest. 95: 1505–1511. 4. Leslie, R. D. G., Atkinson, M. A., and Notkins, A. L. (1999). Autoantigens IA-2 and GAD in type I (insulin-dependent) diabetes. Diabetologia 42: 3–14. 5. Karlsen, A. E., et al. (1991). Cloning and primary structure of a human islet isoform of glutamic acid decarboxylase from chromosome 10. Proc. Natl. Acad. Sci. USA 88: 8337–8341. 6. Bu, D. F., et al. (1992). Two human glutamate decarboxylases, 65-kDa GAD and 67kDa GAD, are each encoded by a single gene. Proc. Natl. Acad. Sci. USA 89: 2115–2119. 7. Kaufman, D. L., Houser, C. R., and Tobin, A. J. (1991). Two forms of the ␥-aminobutyric acid synthetic enzyme glutamate decarboxylase have distinct intraneuronal distributions and cofactor interactions. J. Neurochem. 56: 720–723. 8. Martin, D. L., Martin, S. B., Wu, S. J., and Espina, N. (1991). Regulatory properties of brain glutamate decarboxylase (GAD): the apoenzyme of GAD is present principally as the smaller of two molecular forms of GAD in brain. J. Neurosci. 11: 2725–2731. 9. Faulkner-Jones, B. E., Cram, D. S., Kun, J., and Harrison, L. C. (1993). Localization and quantitation of expression of two glutamate decarboxylase genes in pancreatic -cells and other peripheral tissues of mouse and rat. Endocrinology 133: 2962–2972. 10. Persson, H., et al. (1990). Expression of the neurotransmitter-synthesizing enzyme glu-
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tamic acid decarboxylase in male germ cells. Mol. Cell. Biol. 10: 4701–4711. 11. Menard, V., Jacobs, H., Jun, H. S., Yoon, J. W., and Kim, S. W. (1999). Anti-GAD monoclonal antibody delays the onset of diabetes mellitus in NOD mice. Pharm. Res. 16: 1059–1066. 12. Yoon, J. W., et al. (1999). Control of autoimmune diabetes in NOD mice by GAD expression or suppression in  cells. Science 284: 1183–1187. 13. Zamecnik, P. C., and Stehenson, M. L. (1978). Inhibition of Rous sarcoma virus replication and cell transformation by a specific oligodeoxynucleotide. Proc. Natl. Acad. Sci. USA 75: 280–284. 14. Krystal, G. W., Armstrong, B. C., and Battey, J. F. (1990). N-myc mRNA forms an RNARNA duplex with endogenous antisense transcripts. Mol. Cell. Biol. 10: 4180–4191. 15. Brossalina, E., Pascolo, E., and Toulme, J. J. (1993). The binding of an antisense oligonucleotide to a hairpin structure via triplex formation inhibits chemical and biological reactions. Nucleic Acids Res. 21: 5616–5622. 16. Galderisi, U., Cascino, A., and Giordano, A. (1999). Antisense oligonucleotides as therapeutic agents. J. Cell. Physiol. 181: 251–257. 17. Capaccioli, S., Di Pasquale, G., Mini, E., Mazzei, T., and Quattrone, A. (1993). Cationic lipids improve antisense uptake and prevent degradation in cultured cells and in human serum. Biochem. Biophys. Res. Commun. 197: 818–825. 18. Agrawal, S., and Iyer, R. P. (1995). Modified oligonucleotides as therapeutic and diagnostic agents. Curr. Opin. Biotechnol. 6: 12–19. 19. Galderisi, U., et al. (1999). Antisense inhibitory effects: a comparison between 3⬘-partial and full phosphorothioate antisense oligonucleotides. J. Cell. Biochem. 74: 31–37. 20. Monia, B. P. (1997). First- and second-generation antisense inhibitors targeted to human c-raf kinase: in vitro and in vivo studies. Anticancer Drug Res. 12: 327–339. 21. Agrawal, S., and Zhao, Q. (1998). Antisense therapeutics. Curr. Opin. Chem. Biol. 2: 519–528. 22. Shinozuka, K., Okamoto, T., Matsukura, M., and Sawai, H. (1997). Synthesis and antiHIV activity of phosphorothioate antisense DNA containing C-5 polyamine substituted 2⬘-deoxyuridine and/or acridine residues. Nucleic Acids Symp. Ser. 37: 215–216. 23. Bielinska, A., Kukowska-Latallo, J. F., Johnson, J., Tomalia, D. A., and Baker, J. R., Jr. (1996). Regulation of in vitro gene expression using antisense oligonucleotides or antisense expression plasmids transfected using starburst PAMAM dendrimers. Nucleic Acids Res. 24: 2176–2182. 24. Choi, Y. H., et al. (1998). Polyethylene glycol-grafted poly-L-lysine as polymeric gene carrier. J. Control. Release 54: 39–48.
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25. Xi, M. C., Morales, F. R., and Chase, M. H. (1999). Evidence that wakefulness and REM sleep are controlled by a GABAergic pontine mechanism. J. Neurophysiol. 82: 2015–2019. 26. Frye, C. A., and Vongher, J. M. (1999). GABA(A), D1, and D5, but not progestin receptor, antagonist and anti-sense oligonucleotide infusions to the ventral tegmental area of cycling rats and hamsters attenuate lordosis. Behav. Brain Res. 103: 23–34. 27. Terasawa, E., Luchansky, L. L., Kasuya, E., and Nyberg, C. L. (1999). An increase in glutamate release follows a decrease in ␥ aminobutyric acid and the pubertal increase in luteinizing hormone releasing hormone release in the female rhesus monkeys. J. Neuroendocrinol. 11: 275–282. 28. Kasuya, E., Nyberg, C. L., Mogi, K., and Terasawa, E. (1999). A role of ␥-amino butyric acid (GABA) and glutamate in control of puberty in female rhesus monkeys: effect of an antisense oligodeoxynucleotide for GAD67 messenger ribonucleic acid and MK801 on luteinizing hormone-releasing hormone release. Endocrinology 140: 705–712. 29. Bannai, M., Ichikaw, M., Nishihara, M., and Takahashi, M. (1998). Effect of injection of antisense oligodeoxynucleotides of GAD isozymes into rat ventromedial hypothalamus on food intake and locomotor activity. Brain Res. 784: 305–315. 30. Odagiri, H., Wang, J., and German, M. S. (1996). Function of the human insulin promoter in primary cultured islet cells. J. Biol. Chem. 271: 1909–1915. 31. German, M. S., Moss, L. G., and Rutter, W. J. (1990). Regulation of insulin gene expression by glucose and calcium in transfected primary islet cultures. J. Biol. Chem. 265: 22063–22066. 32. Mitanchez, D., Doiron, B., Chen, R., and Kahn, A. (1997). Glucose-stimulated genes and prospects of gene therapy for type I diabetes. Endocr. Rev. 18: 520–540. 33. Barry, M. E., et al. (1999). Role of endogeneous endonucleases and tissue site in transfection and CpG-mediated immune activation after naked DNA injection. Hum. Gene Ther. 10: 2461–2480. 34. Miyazaki, J., et al. (1990). Establishment of a pancreatic  cell line that retains glucoseinducible insulin secretion: special reference to expressioiin of glucose transporter isoforms. Endocrinology 127: 126–132. 35. Chomczynski, P., and Sacchi, N. (1987). Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162: 156–159. 36. Gottlieb, D. I., Chang, Y. -C., and Schwob, J. E. (1986). Monoclonal antibodies to glutamic acid decarboxylase. Proc. Natl. Acad. Sci. USA 83: 8808–8812. 37. Lacy, P. E., and Kostianovsky, M. (1967). Method for the isolation of intact islets of Langerhans from the rat pancreas. Diabetes 16: 35–39.
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Repression of GAD Autoantigen Expression in Pancreas -Cells by Delivery of Antisense Plasmid/PEG-g-PLL Complex Minhyung Lee,1 Sang-oh Han,1 Kyung Soo Ko,1 Jae Joon Koh,1 Jong-Sang Park,2 Ji-Won Yoon,3 and Sung Wan Kim1,* 1
Center for Controlled Chemical Delivery, Department of Pharmaceutics and Pharmaceutical Chemistry, University of Utah, Salt Lake City, Utah 84112, USA 2 Department of Chemistry, Seoul National University, Seoul 151-742, Korea 3 Julia-Mcfarlane Diabetes Research Center, University of Calgary, Calgary, Alberta, Canada T2N 4N1 *To whom correspondence and reprint requests should be addressed. Fax: (801) 581-7848. E-mail: [email protected].
It was previously reported that silencing of the expression of glutamic acid decarboxylase (GAD) in transgenic nonobese diabetic (NOD) mice completely protected islet -cells against development of diabetes. This suggests that the repression of GAD autoantigen by somatic gene delivery can prevent autoimmune destruction of pancreatic -cells. To repress GAD expression in islet -cells, we delivered an antisense GAD mRNA expression plasmid (pRIP-AS-GAD) using poly(ethylene glycol)-grafted poly-L-lysine (PEG-g-PLL) as a gene carrier. In a gel retardation assay, the pRIP-AS-GAD/PEG-g-PLL complex was completely retarded above a weight ratio of 1:1.5 (plasmid:PEG-g-PLL). PEG-g-PLL protected the plasmid DNA from DNase I for more than 60 minutes. In a reporter gene transfection assay, PEG-g-PLL showed the highest transfection efficiency at a weight ratio of 1:3. We also transfected pRIP-AS-GAD/PEG-g-PLL complex into a GAD-producing mouse insulinoma (MIN6) cell line. The antisense mRNA was expressed specifically in -cells and expression was dependent on glucose level. The repression of GAD after transfection of pRIPAS-GAD was confirmed by immunoblot assay. In addition, in vivo expression of antisense RNA in pancreas was confirmed by RT-PCR after intravenous injection of the complex into mice. Therefore, our study revealed that the pRIP-AS-GAD/PEG-g-PLL system is applicable for the repression of GAD autoantigen expression. Key words: antisense plasmid, autoimmune diabetes, glutamic acid decarboxylase (GAD), gene delivery, pancreatic -cell, polyethylene glycol-grafted poly-L-lysine (PEG-g-PLL)
INTRODUCTION Type 1 (insulin-dependent) diabetes mellitus results from autoimmune destruction of the -cells of the islets of Langerhans. The destructive process is thought to be immune mediated [1]. Glutamic acid decarboxylase (GAD) 65 has been identified as a major autoantigen in type I diabetes, where GAD65-specific antibodies are present in about 80% of newly diagnosed patients [2–4]. GAD catalyzes the decarboxylation of L-glutamic acid to ␥aminobutyric acid (GABA), the major inhibitory neurotransmitter in the mammalian brain. Two isoforms, named GAD65 and GAD67, have been cloned and the human isoforms are approximately 65% identical at the amino acid level [5,6]. They can be distinguished by their molecular masses, their cofactor interactions, and their subcellular distributions [7,8]. GAD65 is largely membranebound and is relatively enriched in vesicular membranes,
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whereas GAD67 is soluble and distributed in the cytoplasm. GAD is expressed primarily in GABAnergic neurons of the adult mammalian central nervous system, but is also present in pancreatic -cells and testes [9,10]. It was reported that anti-GAD monoclonal antibody delays the onset of diabetes mellitus in nonobese diabetic (NOD) mice [11]. In addition, silencing of the expression of GAD65 and GAD67 in the -cells completely protected against development of diabetes in the transgenic NOD mouse, suggesting that GAD has a major role in the development of type 1 diabetes mellitus (IDDM) [12]. These data suggest that the repression of GAD autoantigen by somatic gene delivery of antisense expression plasmid can prevent autoimmune destruction of pancreatic -cells. Delivery of antisense DNA has been widely used to control specific gene expression [13]. The principle of antisense inhibition relies on the specificity of base-pair formation
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FIG. 1. Diagram of pRIP-AS-GAD structure. RIP, rat insulin promoter; I/A, polyadenylation signal.
[14–16]. This specificity enables antisense nucleic acids to inhibit the transcription or translation of specific genes. Synthetic oligonucleotides or antisense expression plasmid has been used for antisense inhibition, but the major limitation of antisense inhibition is that it is difficult to deliver sufficient amounts of antisense DNA for substantial inhibition of the expression of a specific gene. This is due to the inefficiency of naked DNA entry into the cells. Therefore, most antisense studies have used modified DNA structures to increase the availability and the stability of the nucleic acid [17–19]. Phosphorothioate is one of the most frequent variants of antisense oligonucleotides [16,18,20–22]. Cationic lipid or polymer was also used to improve the stability and availability of the nucleic acid [17,23]. Poly(ethylene glycol)-grafted poly-L-lysine (PEG-g-PLL) is a polymeric gene carrier [24] that has lower cytotoxicity and higher transfection efficiency than PLL. Here we synthesize three kinds of PEG-g-PLLs with various PEG contents. We evaluated complex formation and stabilization effects of these PEG-g-PLLs. Antisense expression plasmid/PEG-g-PLL complex was delivered to GAD-producing cells to repress GAD expression. The antisense GAD mRNA expression was driven by the rat insulin gene promoter for cell-specific and glucose-concentration-dependent expression. The transfection conditions were optimized for the delivery of antisense expression plasmid. The expression of antisense GAD mRNA was confirmed by RT-PCR, and GAD protein level is measured by immunoblot assay. In addition, in vivo expression of antisense GAD mRNA in pancreas was confirmed by RT-PCR after the intravenous injection of the complex into mice.
We carried out a gel retardation assay to investigate whether the synthesized PEG-g-PLL forms a complex with the antisense expression plasmid, pRIP-AS-GAD. pRIP-ASGAD consists of GAD65 and GAD67 cDNAs in the antisense direction, the rat insulin gene promoter, and polyadenylation signals (Fig. 1). The band of plasmid DNA was retarded as the amount of PEG-g-PLL increased, suggesting that PEG-g-PLL forms a complex with plasmid DNA (Fig. 2). The plasmid band was completely retarded above a 1:1.5 weight ratio of plasmid DNA:PEG-g-PLL (Fig. 2, lanes 4–7). A DNase I protection assay was carried out to confirm that PEG-g-PLL protected plasmid DNA from DNase I. The protection efficiencies of 10, 15, and 20 mole percent (mol%) PEG-g-PLL were evaluated. After complex formation at a 1:1.5 weight ratio (plasmid DNA:PEG-g-PLL), the plasmid DNA/PEG-g-PLL complexes were incubated with DNase I for 20 or 60 minutes. Naked DNA was completely degraded by DNase I after 20 minutes of incubation (Fig. 3, lanes 2 and 3), but 10 mol% PEG-g-PLL protected plasmid DNA over 60 minutes (Fig. 3, lanes 4 and 5). We found that 15 and 20 mol% PEG-g-PLLs were less effective than 10 mol% PEG-g-PLL in protection (Fig. 3, lanes 6–9). Transfection Assay with pSV--galactosidase/PEG-gPLL Complex To determine the most effective weight ratio of plasmid DNA:polymer, a reporter gene transfection assay was carried out. We used pSV--galactosidase as a reporter gene for monitoring gene expression in 293T cells. In pSV-galactosidase, the expression of lacZ (encoding -galactosidase) was driven by SV40 early promoter and enhancer. Various plasmid DNA/polymer complexes were formulated with a fixed amount of plasmid DNA (20 g) and increasing amounts of 10 mol% PEG-g-PLL (20–120 g). A 1:3
RESULTS Synthesis and Characterization of PEG-g-PLL We synthesized three kinds of PEG-g-PLLs with various PEG contents and characterized them by 1H-NMR. The content of PEG was calculated from the 1H-NMR spectrum.
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FIG. 2. Gel retardation assay. pRIP-AS-GAD plasmid (1 g) was mixed with various quantities of PEG-g-PLL. After incubation at room temperature for 30 min, the complexes were analyzed by 0.7% agarose gel electrophoresis. Lane 1, plasmid-only control.
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FIG. 3. DNase I protection assay. pRIP-AS-GAD/PEG-g-PLL complex solution was incubated with DNase I for 20 min or 60 min. After incubation, DNA was analyzed by 0.7% agarose gel electrophoresis.
Expression of GAD in MIN6 cells was also confirmed by an immunoblot assay. Cell extracts from MIN6 cells or BALB/c mouse islets were separated in 10% polyacrylamide gel and analyzed with anti-GAD antibody. Although the expression level of GAD in MIN6 cell extract was lower than that in mouse islet extract, GAD65 and GAD67 were detected in both extracts (Fig. 6B, lane 1 and 2). Therefore, MIN6 cells can be used as an in vitro GAD-producing cell model.
weight ratio of plasmid DNA:PEG-g-PLL showed the most effective transfection (Fig. 4). This result was exactly the same as in a previous report [24]. In this previous report, in which 5, 10, and 25 mol% PEG-g-PLLs were used, three kinds of PEG-g-PLLs showed little difference in complex formation ability above a 1:3 weight ratio (plasmid DNA:PEG-g-PLL) [24]. In addition, 10 mol% PEG-g-PLL showed the highest transfection efficiency at a 1:3 weight ratio (plasmid DNA:PEG-g-PLL). Therefore, we carried out the transfection assay with 10, 15, and 20 mol% PEG-g-PLL at a 1:3 weight ratio (plasmid DNA:PEG-g-PLL) to determine which PEG-g-PLL was the most effective in transfection. The complexes were transfected into MIN6 cells. Among those tested, the 10 and 15 mol% PEG-g-PLL showed almost the same transfection efficiency (Fig. 5), but 20 mol% PEG-g-PLL was less effective than 10 and 15 mol% PEG-g-PLL. Therefore, we used 10 mol% PEG-g-PLL in the following experiments, because it protected plasmid DNA from DNase I most effectively and showed high transfection efficiency.
Antisense GAD mRNA Expression in MIN6 Cells We transfected pRIP-AS-GAD/PEG-g-PLL complex into MIN6 cells and carried out RT-PCR to detect the antisense GAD67 mRNA. We mixed 5, 10, or 20 g of pRIP-AS-GAD plasmid with PEG-g-PLL at a weight ratio of 1:3 and transfected into MIN6 cells. At 48 hours after transfection, the cells were collected and total RNA was prepared. To determine the expression level, antisense GAD67 mRNA was amplified. We identified a 357-bp PCR product in agarose gel electrophoresis. The level of antisense GAD67 mRNA increased with increasing amounts of plasmid (Fig. 7A). Duration of antisense GAD mRNA expression was also evaluated by RT-PCR (Fig. 7B). Although the expression level of antisense GAD mRNA decreased, antisense GAD mRNA was detected in the cells at 96 hours after transfection. Antisense mRNA expression was driven by the rat insulin gene promoter. Therefore, the antisense GAD mRNA expression was tissue (pancreas islet)-specific and responsive to glucose concentration. To confirm the tissuespecific expression, pRIP-AS-GAD/PEG-g-PLL complexes were transfected to MIN6 cells or 293T cells and the
GAD Expression in MIN6 Cells Before using MIN6 cells as an in vitro GAD-producing cell model, we confirmed by RT-PCR that MIN6 cells produce GAD antigen. Total RNA was prepared from MIN6 cells and used as a template. We identified 293-bp and 357bp PCR products, which are the expected sizes for GAD65 and GAD67 mRNAs, respectively. This result suggested that GAD65 and GAD67 were produced in MIN6 cells (Fig. 6A, lanes 1–4). -Actin mRNA was amplified as an endogeneous control (Fig. 6A, lanes 5 and 6).
FIG. 4. Effect of plasmid DNA:PEG-g-PLL weight ratio on transfection of 293T cells. DNA/PEG-g-PLL complexes were formulated with a fixed amount of plasmid DNA (20 g) and increasing amounts of 10 mol% PEG-g-PLL (20–120 g). After 30 min of incubation at room temperature, the complexes were transfected to 293T cells. Transfection efficiency was measured by -galactosidase assay. Bar, standard error of mean.
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FIG. 5. Effect of PEG contents in PEG-g-PLL on transfection efficiency of MIN6 cells. DNA/PEG-g-PLL complexes were formulated with plasmid DNA (20 g) and polymer (60 g). After 30 min of incubation at room temperature, the complexes were transfected to MIN6 cells. Transfection efficiency was measured by -galactosidase assay. Bar, standard error of mean.
the transfected cells. We transfected 5, 10, or 20 g pRIPAS-GAD into MIN6 cells by formation of complexes with PEG-g-PLL. At 48 hours after transfection, the cell extracts were prepared and analyzed with anti-GAD antibody. The expression levels of GAD65 and GAD67 decreased significantly with increasing amounts of pRIP-AS-GAD (Fig. 8).
expressed antisense GAD mRNAs were amplified by RTPCR. We detected a PCR product of 357 bp for antisense GAD67 mRNA in RT-PCR with total RNA from MIN6 cells (Fig. 7C, lane 3). However, RT-PCR with total RNA from 293T cells did not show any signal (Fig. 7C, lane 2). To confirm the glucose concentration responsive expression of antisense GAD mRNA, the transfected cells were maintained in medium containing 0, 1, 5, or 25 mM glucose. The expression level of antisense GAD mRNA was measured by RT-PCR (Fig. 7D), revealing that the expression level increased as the glucose level increased in the medium. Therefore, antisense GAD mRNA is expressed in response to the glucose level in the medium.
In Vivo Expression of Antisense mRNA in Mouse Pancreas after Injection of pRIP-AS-GAD/PEG-g-PLL Complex To evaluate the expression of antisense GAD mRNA in mouse pancreas, pRIP-AS-GAD/PEG-g-PLL complex was injected intravenously into mice. PEG-g-PLL was injected into mice as a control. To detect antisense GAD mRNA, RTPCR was carried out with total RNA prepared from the pancreas. Antisense RNA was detected in the pancreas of mice injected with pRIP-AS-GAD/PEG-g-PLL complex after 1 or 3 days of injection (Fig. 9). However, in the pancreas of mice injected with PEG-g-PLL only, antisense RNA was not detected. These results suggest that the pRIP-ASGAD/PEG-g-PLL complex is applicable to in vivo repression of GAD antigen.
DISCUSSION
Repression of GAD in MIN6 Cells by Transfection of pRIP-AS-GAD/PEG-g-PLL Complex To evaluate the repression of GAD in MIN6 cells, an immunoblot assay was carried out with cell extracts from
Antisense repression of GAD has been tried by several researchers on the field of the nervous system [25–29]. These studies were designed to evaluate the effect of the changes of GABA and glutamate concentrations on the differentiation of the nervous system, food intake, or locomotive behavior. In all of these studies, antisense oligonucleotides were used. Also, it was reported that
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FIG. 6. Expression of GAD in MIN6 cells. (A) Detection of GAD65, GAD67, and -actin mRNAs in MIN6 cells by RT-PCR. Total RNA was prepared from MIN6 cells. Total RNA was reverse transcribed and amplified by PCR using specific primers. Lanes 1 and 2, GAD67; lanes 3 and 4, GAD65; lanes 5 and 6, -actin. M, DNA size marker. (B) Detection of GAD65 and GAD67 in mouse islet and MIN6 cells by immunoblot assay. Cell extracts (40 g) were separated in 10% SDS-polyacrylamide gel and transferred to PVDF membrane. GAD65 and GAD67 were detected by immunoblot assay.
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FIG. 7. Expression of antisense GAD mRNA in MIN6 cells after transfection. (A) Dose-dependent antisense GAD mRNA expression. Total RNA was prepared from transfected MIN6 cells. GAD67 mRNA was reverse transcribed and amplified by PCR using specific primers. M, DNA size marker. (B) Duration of antisense GAD mRNA expression. Total RNA was prepared from transfected MIN6 cells at the indicated time and analyzed by RT-PCR. M, DNA size marker. (C) Cell-type-specific antisense GAD mRNA expression. pRIP-AS-GAD/PEG-gPLL complexes were transfected to 293T cells (lane 2) or MIN6 cells (lane 3). Total RNA was prepared from 293T cells or MIN6 cells and analyzed by RTPCR. M, DNA size marker. (D) Glucose-responsive antisense GAD mRNA expression. MIN6 cells were cultured in glucose-containing medium as indicated. At 48 h after transfection, total RNA was prepared and analyzed by RTPCR. M, DNA size marker; lane 1, negative control without transfection.
silencing of the expression of GAD in transgenic NOD mice with an antisense GAD transgene completely protected islet -cells against the development of autoimmune diabetes [12]. However, somatic gene delivery of antisense expression plasmid for prevention of type 1 diabetes has not been reported. In our study, pRIP-AS-GAD plasmid instead of antisense oligonucleotides was delivered to GAD-producing MIN6 cells by complex formation with PEG-g-PLL. This pRIP-AS-GAD/PEG-g-PLL system has advantages over modified antisense oligonucleotides. Unlike the antisense oligonucleotide approach, this system allows delivery of a larger size of DNA using PEG-g-PLL as a gene carrier. The antisense oligonucleotide system uses 15–25 bp oligonucleotides. Therefore, the oligonucleotides should be designed to be complementary to the most effective site for inhibition of GAD expression. However, in the pRIP-AS-GAD/PEG-g-PLL system, whole antisense cDNA was delivered and, therefore, antisense mRNA can hybridize with the sense mRNA effectively. In addition, the
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delivery of larger sizes of DNA allowed the insertion of the rat insulin gene promoter into the plasmid. As a result, the antisense mRNA was expressed specifically in pancreas. Also, the insulin gene promoter has been reported to respond to the glucose level [30–32], and we confirmed that expression of antisense GAD mRNA was induced by increasing glucose concentration (Fig. 7D). We synthesized three kinds of PEG-g-PLL and characterized them to optimize the antisense plasmid transfection. The protective effect of PEG-g-PLL was evaluated by DNase I protection assay (Fig. 3). Previously, DNase I was demonstrated as one of many nucleases existing in serum [33]. Therefore, the stability of the plasmids in the presence of DNase I is a critical parameter for in vivo gene delivery. After DNase I treatment, 10 mol% PEG-g-PLL protected the plasmid DNA most effectively (Fig. 3); 15 or 20 mol% PEG-g-PLL was less effective in protection of DNA. It was expected that higher mol% PEG-g-PLLs would require a higher plasmid DNA:polymer ratio to achieve effective DNA protection.
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FIG. 8. Repression of GAD antigen expression. At 48 h after transfection, cell extracts were prepared from the transfected cells. Extracts were separated in 10% SDS-polyacrylamide gel and transferred to PVDF membrane. GAD67 and GAD65 were detected by immunoblot assay.
We found that 10 mol% PEG-g-PLL showed the highest transfection efficiency at a weight ratio of 1:3 (plasmid DNA:PEG-g-PLL) in a reporter gene transfection assay (Fig. 4). At this weight ratio, pRIP-AS-GAD/PEG-g-PLL complex was formed and transfected to MIN6 cells. It has not been reported that MIN6 cells produce GAD autoantigen. However, we confirmed the expression of GAD in MIN6 cells by RT-PCR and immunoblot assay (Figs. 6A and 6B). Therefore, MIN6 cells are a good in vitro GAD-producing cell model for evaluation of GAD repression. pRIP-ASGAD/PEG-g-PLL expressed antisense GAD mRNA over 96 hours in MIN6 cells and repressed the expression of GAD antigen at a substantial level. After the injection, the expression of antisense mRNA was evaluated by RT-PCR. The antisense mRNA was expressed in the pancreas of mice injected with pRIP-ASGAD/PEG-g-PLL complex. The antisense RNA was expressed for more than 3 days, suggesting that pRIP-ASGAD/PEG-g-PLL complex is applicable to in vivo repression of GAD antigen. A previous report showed that the pancreatic insulin content and plasma insulin concentrations of RIP-AS-GAD transgenic mice were indistinguishable from those of transgene negative NOD mice [12]. This suggests that repression of GAD expression does not have any side effects on normal function of pancreas. Also, GAD expression in brain was detected equally in brain tissue of transgene-negative NOD mice and RIP-AS-GAD transgenic NOD mice, suggesting that the repression of GAD by pRIP-ASGAD/PEG-g-PLL may not have any effect on normal expression of GAD in other organs [12]. Therefore, the pRIP-AS-GAD/PEG-g-PLL system is effective to repress the expression of GAD autoantigen specifically in pancreatic -cells.
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FIG. 9. Expression of antisense GAD mRNA in pancreas of mice after the intravenous injection of pRIP-AS-GAD/PEG-g-PLL complex. Total RNAs were prepared from the pancreas of the mice injected with pRIP-AS-GAD/PEG-g-PLL complex or PEG-g-PLL only. GAD67 mRNA was reverse transcribed and amplified by PCR using specific primers. M, DNA size marker.
MATERIALS
AND METHODS
Preparation of pRIP-AS-GAD. The construction of the antisense GAD expression plasmid has been described [12]. The antisense expression plasmid, pRIP-AS-GAD, consists of GAD65 and GAD67 cDNAs in the antisense direction, the rat insulin gene promoter, and polyadenylation signals (Fig. 1). The expression of antisense mRNA was driven by the rat insulin gene promoter. pRIP-AS-GAD was introduced into Escherichia coli strain JM109 and isolated in large quantities using the plasmid purification Maxi kit (Qiagen, Valencia, CA). Purity of plasmids was certified by OD260/OD280 ratio and by distinctive bands of DNA fragments at corresponding base pairs in agarose gel electrophoresis following XhoI and NotI digestion. The concentration of plasmid DNA was determined using OD260 = 50 g of DNA. The plasmid DNA was stored at –20⬚C until use. Synthesis of PEG-g-PLL. We prepared three different types of PEG-g-PLL with various PEG contents of 10, 15, and 20 mol% as described [24] with slight modification. Synthesis of 10 mol% PEG-g-PLL is described as an example. PLL-hydrobromide (100 mg) was dissolved in 1 ml DMSO and 10 l TEA and stirred for 30 min. M-SPA-2000 (100 mg) was dissolved in 2 ml DMSO and added into PLL solution for 30 min. The mixed solution was stirred at room temperature for 6 h. The resulting material was precipitated in excess of ethylether. The material was washed with ether three times and then with methylene chloride three times. The precipitated polymer was dried and dissolved in 2 ml deionized water. The synthesized PEG-g-PLL was purified by dialysis against deionized water, followed by lyophilization. The polymer was characterized the contents of PEG by 1H-NMR in D2O. Gel retardation assay. Increasing amounts of PEG-g-PLL, ranging from 0.5 to 3 g, were added to 1 g pRIP-AS-GAD plasmid DNA. The mixtures were incubated at room temperature for 30 min and electrophoresed on 0.7% (w/v) agarose gel. DNase I protection assay. The weight ratio of plasmid/PEG-g-PLL complex was 1:3 (plasmid:PEG-g-PLL) in phosphate buffered saline (PBS). After complex formation, DNase I (10 units, Gibco BRL, Gaithersburg, MD) was added to the complex solution and the reaction mixture was incubated at 37⬚C. The sample (50 l) was taken at 20 or 60 min after incubation, mixed with 50 l of 2⫻ stop solution (0.4 M NaCl, 80 mM EDTA, and 2% SDS), and placed on ice. To dissociate the plasmid DNA from PEG-g-PLL, the mixtures were incubated at 60⬚C overnight. After phenol/chloroform extraction, the DNA was precipitated with ethanol. The pellets were dissolved in 10 l TrisEDTA (TE) buffer and applied to the 0.7% agarose gel electrophoresis.
MOLECULAR THERAPY Vol. 4, No. 4, October 2001 Copyright © The American Society of Gene Therapy
doi:10.1006/mthe.2001.0458, available online at http://www.idealibrary.com on IDEAL
Cell culture. MIN6 cells and 293T cells were maintained in DMEM supplemented with 15% (MIN6 cells) or 10% (293T cells) FBS in a 5% CO2 incubator. MIN6 cells retain glucose-inducible insulin secretion like normal cells [34]. For the transfection studies, MIN6 or 293T cells were seeded at a density of 2.0 ⫻ 106 cells/100-mm culture dish and incubated for 24 h before the addition of the plasmid DNA/polymer complex. Transfection. The in vitro transfection assay was carried out as follows. Plasmid DNA/PEG-g-PLL complexes were prepared by mixing 20 g of plasmid DNA and various amounts of PEG-g-PLL (20, 40, 60, or 120 g) in 500 l of serum-free DMEM medium, and incubated for 30 min at room temperature. The MIN6 or 293T cells were washed twice with serum-free DMEM medium, then 10 ml fresh serum-free medium was added. Plasmid DNA/PEG-g-PLL (500 l) complex was added to each dish. The cells were then incubated for 4 h at 37⬚C in a 5% CO2 incubator. After 4 h, the transfection mixtures were removed and 10 ml of fresh DMEM medium containing FBS was added to each dish. Cells were incubated for a desired time at 37⬚C. -Galactosidase assay. Cells in each dish were washed twice with PBS and 900 l of 1⫻ reporter lysis buffer (Promega, Madison, WI) was added to cover the cells. After 15 min of incubation at room temperature, the cells were collected and transferred to a microcentrifuge tube. After 15 s of vortexing, the cells were centrifuged at 12,000g for 2 min. The extracts were transferred to a fresh tube and stored at –70⬚C until use. The protein concentrations of the extracts were determined using the BCA protein assay kit (Pierce, Rockford, IL). -Galactosidase assay mixtures included 100 g cell extracts and 150 l of assay 2⫻ buffer (Promega, Madison, WI) in 300 l of volume. The mixtures were incubated at 37⬚C for 90 min. After the reaction, 500 l of 1 M sodium carbonate was added to the mixtures and the absorbance at 420 nm was measured. RT-PCR. The transfected cells were washed twice with PBS and total RNA was prepared by acid-guanidium thiocyanate-phenol-chloroform extraction as described [35], using RNAwiz (Ambion, Austin, TX). The total RNA was treated with RNase-free DNase I (Promega, Madison, WI) to eliminate the contaminated DNA. The concentration of RNA was measured by the absorbance at 260 nm, and the integrity of RNA was confirmed by formaldehyde-formamide denatured agarose gel electrophoresis. Total RNA (2 g) was hybridized to the backward primer and reverse transcribed using AMV reverse transcriptase (Promega, Madison, WI). The reverse-transcribed samples were amplified by PCR using Taq polymerase (Life Promega, Madison, WI). The sequences of the specific oligonucleotide primers were as follows: GAD67 forward primer, 5⬘-ATGACGTCTCCTACGATACA-3⬘; GAD67 backward primer, 5⬘-CCCCTTGAGGCTGGTAACCA-3⬘; GAD65 forward primer, 5⬘-AAGGGGACTACTGGATTTGA-3⬘; GAD65 backward primer, 5⬘-TGCGGAAGAAGTTGACCTTA-3⬘; -actin forward primer, 5⬘-TGGAATCCTGTGGCATCCATGAAA C-3⬘; -actin backward primer, 5⬘TAAAACGCAGCTCAGTAACAGTCCG-3⬘. The PCR reaction consisted of 94⬚C for 3 min, 35 cycles at 94⬚C for 30 s, 58⬚C for 30 s, and 72⬚C for 1 min, followed by an extension of 10 min at 72⬚C. The PCR products were separated by electrophoresis in 2% agarose gels. The lengths of the expected products were 357 bp for GAD67, 293 bp for GAD65, and 354 bp for -actin. Anti-GAD antibody production and purification. A hybridoma cell line producing anti-GAD antibody (HB184, IgG1, anti-GADF-6 that is GAD65 and GAD67 specific) was obtained from ATCC (Rockville, MD). The hybridoma cell line was injected into the peritoneum of Balb/c mice to produce anti-GAD antibody in the ascites fluid. Anti-GAD antibody was extracted and purified after precipitation with saturated ammonium sulfate, followed by anion exchange chromatography and protein A-Sepharose chromatography, as described [36]. The purified fractions were pooled, dialyzed, and filtered with 0.2-m sterile Acrodisk membranes, diluted to a concentration of 2 mg/ml in PBS, and stored at –20⬚C. The purified fractions were characterized by electrophoresis (Phastgel 8/25, Pharmacia, Piscataway, NJ) under native and denaturing conditions, and with a homogeneous gel in denaturing and reducing conditions. Islet isolation. Islets of Langerhans were isolated from male Balb/c mouse pancreas by a modified collagenase digestion technique and discontinuous Ficoll density gradient centrifugation [37]. Briefly, the pancreas was removed after swelling by collagenase solution injection (5–7 ml/pancreas,
MOLECULAR THERAPY Vol. 4, No. 4, October 2001 Copyright © The American Society of Gene Therapy
ARTICLE
1 mg/ml) through the common bile duct and incubated for 15 min at 37⬚C. The digested fragments of the pancreas were collected and washed with HBSS. Finally, the acinar cells and islets were separated by Ficoll density gradient centrifugation. The islet-rich layer was collected and washed with HBSS, and islets were suspended in RPMI-1640 medium with 10% FBS and incubated at 37⬚C under humidified conditions with 5% CO2. On average 100–150 islets were isolated from a mouse pancreas. Immunoblot assays. The cells were washed with PBS twice and 1.0 ml lysis buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.02% sodium azide, 100 g/ml phenylmethanesulfonic fluoride (PMSF), 1 g/ml aprotinin, and 1% Nonidet P-40) was added to each dish. The cells were incubated for 20 min on ice. The cells were harvested and transferred to a new tube. For the preparation of cell extracts from pancreas, pancreas was homogenized in lysis buffer and transferred to a new tube. After the centrifugation at 12,000g for 2 min at 4⬚C, the extracts were transferred to a new tube. The extracts were stored at –70⬚C until use. The protein concentrations of the extracts were determined by using BCA protein assay kit (Pierce, Rockford, IL). Cell extracts (40 g) from transfected cells were subjected to electrophoresis in a 10% SDS-polyacrylamide gel. For western blot analysis, proteins were transferred to a polyvinylidene difluoride membrane (PVDF, Millipore, Bradford, MA) for 60 min at 300 mA in transfer buffer (10% methanol, 0.01 M 2-[cyclohexylamino]-1-propane-sulfonic acid (CAPS), pH 11). The membrane was washed with PBST (0.01% Tween 20 in PBS) and blocked overnight in blocking buffer (5% non-fat dried milk in PBS). The membrane was incubated in primary antibody solution for 90 min, and then in secondary antibody solution (anti-mouse IgG, Sigma, St. Louis, MO) for 30 min. The bands were visualized by amplified Opti-4CN detection kit (Bio-Rad, Hercules, CA) as described in the manufacturer’s manual. In vivo expression of GAD mRNA in pancreas of mice. pRIP-ASGAD/PEG-g-PLL complexes were prepared at a weight ratio of 1:3 (plasmid:PEG-g-PLL). pRIP-AS-GAD/PEG-g-PLL complexes (200 l) were injected into the tail vein of Balb/c mice at a dose of 50 g DNA per mouse. At designated times, the animals were killed by cervical dislocation and the pancreas was harvested. The expression of antisense GAD mRNA was evaluated by RT-PCR as described above.
ACKNOWLEDGMENTS We thank Jun-Ichi Miyazaki (Osaka University Medical School, Osaka, Japan) for MIN6 cells, Troy Koch (University of Utah) for technical assistance, and Expression Genetics, Inc., for financial support. This work was supported by National Institutes of Health grant DK51689. RECEIVED FOR PUBLICATION JANUARY 19; ACCEPTED JULY 23, 2001.
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