Antisense Oligonucleotide Induced Dystrophin Exon 45 Skipping at a Low Half-Maximal Effective Concentration in a Cell-Free Splicing System

Abstract

Antisense oligonucleotides (AOs) can facilitate the expression of internally deleted dystrophin in dystrophin-deficient Duchenne muscular dystrophy (DMD) by correcting the reading frame of the pre-mRNA with AO-mediated exon skipping. An antisense 18-mer 2′-O-methyl RNA/ethylene-bridged nucleic acid chimera AO targeting exon 45 of the dystrophin gene, AO85, can induce exon 45 skipping efficiently in cultured cells. AO85 is expected to facilitate dystrophin expression in 8%–9% of all DMD patients. Here, we examined the kinetics of AO85-mediated exon 45 skipping in a cell-free splicing system. In vitro transcribed pre-mRNAs containing dystrophin exon 45 and part of its flanking introns within a hybrid minigene were incubated with HeLa cell nuclear extract, and the resultant mRNAs were amplified by semiquantitative reverse transcriptase–polymerase chain reaction. Time-course analysis revealed that the splicing process fitted well to first order kinetics. Addition of AO85 produced an extra spliced product, deleting exon 45 (Δexon 45), indicating AO85-mediated exon 45 skipping. Production of Δexon 45 increased linearly with increasing concentrations of AO85, reaching a maximum of nearly 80% of the transcripts. The half-maximal effective concentration (EC₅₀) of AO85 was 58.0 nM. The percentage of Δexon 45 among the transcripts decreased inversely with the pre-mRNA concentration; Lineweaver-Burk plotting revealed a competitive fashion of AO85 action. The low EC₅₀ indicates high potential of AO85 for clinical application.

Introduction

Antisense oligonucleotides (AOs) are traditionally employed to destroy the target RNA by RNAase H-mediated digestion (Pan and Clawson, 2006). AO action is dependent on binding to specific mRNA sequences through Watson-Crick base pair hybridization, before digestion of the bound fragment by RNAase H. AOs have been applied to the treatment of infectious diseases or cancers by degrading target mRNAs (VAN AERSCHOT, 2006; Bennett and Swayze, 2010), and their kinetics have been studied (Duan et al., 2008). Currently, AOs are attracting much attention as modulators of splicing that can generate new gene transcripts (Perez et al., 2010; Wood et al., 2010).

Duchenne muscular dystrophy (DMD) is a rapidly progressive muscle wasting disease that usually results in death during the third decade. DMD is characterized by a dystrophin deficiency that usually arises from out-of-frame deletion mutations in the dystrophin gene that create a premature stop codon in the dystrophin mRNA. AO-mediated exon skipping therapy has been proposed for the treatment of DMD, producing in-frame dystrophin mRNA from the out-of-frame mRNA by inducing exon skipping. The newly generated in-frame dystrophin mRNA is expected to produce semifunctional, internally deleted dystrophin protein (Takeshima et al., 1995). The production of internally deleted dystrophin has been clinically demonstrated in some patients who showed natural skipping of an exon encoding a nonsense mutation (Shiga et al., 1997; Flanigan et al., 2011). Compared with gene replacement therapy, the benefits of exon skipping with AOs include the ability to use the endogenous gene and easy chemical synthesis of AOs. Currently, induction of exon skipping with AOs is considered one of the most plausible treatments for DMD (AARTSMA-RUS, 2010; Lu et al., 2011). Remarkably, systemic administration of 2′-O-methyl phosphorothioate AO (PRO051) showed a promising result of a modest improvement in the 6 minutes walk test after 12 weeks of the treatment (Goemans et al., 2011).

AOs with phosphorothioate backbones directed against the splicing enhancer sequence within human dystrophin exon 19 provided the first demonstration of exon skipping in human cells (Pramono et al., 1996). Since then, intravenous infusion of AOs against exon 19 has been reported to induce exon 19 skipping and the expression of internally deleted dystrophin in an exon 20-deleted DMD patient (Takeshima et al., 2006). Several chemical modifications of the first-generation phosphorothioate AOs have been introduced over the past decade to improve the efficiency of antisense therapeutics. Morpholino AOs have been shown to induce dystrophin exon 51 skipping and promote dystrophin expression in DMD muscle cells (PARTRIDGE, 2010).

A modified nucleic acid, 2′-O,4′-C-ethylene-bridged nucleic acid (ENA), has high binding affinity for the complementary RNA strand and more nuclease resistance than unmodified nucleic acid (Morita et al., 2001; Veedu and Wengel, 2010). One antisense 2′-O-methyl RNA/ENA (RNA/ENA) chimera was shown to be 40 times more effective than the conventional phosphorothioate backbone oligonucleotides in inducing exon 19 skipping (Yagi et al., 2004). Furthermore, an RNA/ENA chimera against dystrophin exon 41 encoding a nonsense mutation has been shown to induce efficient skipping of a mutated exon 41 (Surono et al., 2004). Considering that skipping of exon 45 is expected to express internally deleted dystrophin in 8%–9% of DMD patients (Aartsma-Rus et al., 2009a; Takeshima et al., 2010), AOs that induce exon 45 skipping have been proposed as one of the most important AOs for DMD treatment. In our previous studies, we demonstrated that an 18-mer RNA/ENA chimera, AO85, strongly induced exon 45 skipping in cultured myocytes (Takagi et al., 2004a; Takeshima et al., 2011). However, its kinetics, including the half-maximal effective concentration (EC50), are still unknown, as for other AOs designed for DMD treatment (AARTSMA-RUS, 2010).

To facilitate the clinical application of AO85, here we examined AO-mediated exon 45 skipping in a cell-free splicing system and determined the splicing parameters of AO85, revealing a low EC₅₀.

Materials and Methods

AO85

An 18-mer RNA/ENA chimera (5′-CgCTgcCCaaTgCCatCC-3′; upper and lower case letters represent ENA and 2′-O-methyl RNA, respectively), AO85, complementary to the 5′ region of dystrophin exon 45 (Takeshima et al., 2011), was synthesized by KNC Laboratories Co. (Kobe, Japan) and dissolved in water.

In vitro transcription

The region encompassing dystrophin exon 45 and part of its flanking introns was amplified from human genomic DNA by polymerase chain reaction (PCR) using a set of primers containing NheI and BamHI restriction enzyme recognition sites (forward primer, Int44FNheI 5′-GCGGCTAGCGCTAACCGAGAGGGTGCTTTT-3′; reverse primer, Int45RBamHI 5′-GCGGGATCCGCAGAAAACCACTAACTAGCCACA-3′; restriction sites underlined). The amplified product was digested with NheI and BamHI (New England Biolabs, Hitchen, UK) and inserted between the 2 exons A and B of the predigested expression vector H492 (Habara et al., 2008) to generate a hybrid minigene (Fig. 1a). The sequence of the hybrid minigene was confirmed by DNA sequencing using an ABI 3130 genetic analyzer (Applied Biosystems, Foster City, CA). A region of the hybrid minigene including exon A, dystrophin exon 45 and exon B was PCR amplified using forward (T7(19), 5′-TAATACGACTCACTATAGG-3′) and reverse (YH304, 5′-CTCGAGCAGCCAGTTAAGTCTCTCAC-3′) primers and then purified using a MinElute PCR Purification kit (QIAGEN, Hilden, Germany). The purified template DNAs were subjected to in vitro transcription using an mMESSAGE mMACHINE High Yield Capped RNA Transcription kit (Ambion, Austin, TX). After treatment of the transcription product with DNase (Ambion), the resultant RNAs were purified on a G-50 Quick Spin Column (Roche Applied Science, Indianapolis, IN) and used as a substrate for the splicing reaction. The concentration and size of the purified RNAs were determined using an ND-1000 spectrophotometer (NanoDrop, Wilmington, DE) and an Agilent 2100 Bioanalyzer with an RNA Nano kit (Agilent Technologies, Santa Clara, CA), respectively.

FIG. 1.

Cell-free splicing reaction. (a) Schematic description of the hybrid-minigene. Pre-mRNA was in vitro transcribed from template DNA that was obtained by PCR amplification of the hybrid-minigene with primers T7 and YH304. Shaded boxes and bars indicate exons and introns, respectively. Dystrophin exon 45 (176 nt) and its flanking introns (222 nt upstream and 210 nt downstream; bold lines) were inserted into the multicloning site of the pre-constructed expression vector H492 that contains exons A and B, a cytomegalovirus (CMV) enhancer-promoter, a T7 promoter, and the bovine growth hormone gene (BGH) polyadenylation signal (boxes). (b) Products of the cell-free splicing reaction. The synthesized pre-mRNA was incubated with HeLa cell nuclear extract for 2 hours at 30°C. The reaction products were RT-PCR amplified and separated by agarose gel electrophoresis (indicated). The structure of each product is described schematically on the right. Boxes and bars indicate exons and introns, respectively. RT-PCR, reverse transcriptase–polymerase chain reaction.

In vitro splicing reaction

The in vitro splicing reaction was carried out at 30°C for 2 hours in a volume of 20 μL containing 30 ng pre-mRNA, 50% (v/v) HeLa cell nuclear extract (Lot No. 4168HNE; Computer Cell Culture Center, Mons, Belgium), 1.6 mM MgCl₂, 0.5 mM ATP, 20 mM creatine phosphate, and 20 U of RNaseOUT (Invitrogen, Carlsbad, CA) (Habara et al., 2008). In some experiments, the indicated amounts of AO85 were added directly to the reaction mixture. The incubation temperature or time was varied to study the temperature or time dependency of the in vitro splicing.

After the designated incubation time, reaction mixtures were treated with Proteinase K (Sigma-Aldrich, St. Louis, MO) and the resulting RNAs were purified by phenol–chloroform extraction and ethanol precipitation as previously described (Habara et al., 2008). Purified total RNA (750 ng) was reverse transcribed using random hexamer primers with M-MLV reverse transcriptase (Invitrogen) according to the manufacturer's protocol and then PCR amplified using a forward primer from exon A (YH307, 5′-ATTACTCGCTCAGAAGCTGTGTTGC-3′) and a reverse primer from exon B (YH308, 5′-AAGTCTCTCACTTAGCAACTGGCAG-3′) (Habara et al., 2008). PCRs were performed in a volume of 20 μL containing 4 μL of cDNA, 1× Ex Taq Buffer, 250 nM dNTPs (Takara Bio, Inc., Kyoto, Japan), 10 pmol of each primer, and 1 U of Ex Taq Polymerase (Takara Bio, Inc.). The PCR cycling conditions were as follows: initial denaturation at 94°C for 2 minutes followed by 16 cycles of denaturation at 94°C for 1 minute, annealing at 58°C for 1 minute, extension at 72°C for 2 minutes, and a final extension at 72°C for 5 minutes. PCR products were analyzed by agarose gel electrophoresis. Each amplified product was semiquantified by measuring the peak areas of capillary electrophoresis using an Agilent 2100 Bioanalyzer with a DNA1000 kit (Agilent Technologies). The sequences of all the detected bands were confirmed by subcloning and sequencing as previously described (Tran et al., 2006). As an internal standard, the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene was also amplified by reverse transcriptase (RT)-PCR from cDNA, using primers spanning exons 3–6 of the GAPDH cDNA (Zhu et al., 2007).

The percentage of spliced products among the total transcripts was calculated by the following formula: splicing efficiency (%)=[spliced/(spliced+unspliced pre-mRNAs)]×100 (Zheng and Baker, 2000). The percentage of the skipped exon among the total transcripts was defined as [Δexon45/(normally spliced product+Δexon45)]×100. The ratio of normally spliced mRNA to the internal standard GAPDH was calculated and its relationship with the pre-mRNA was analyzed.

Statistical analysis

All analyses were performed using GraphPad Prism 5 (GraphPad Software, Inc., San Diego, CA). The mean±standard deviation was derived from 2 or more independent experiments. Both nonlinear and linear regressions were used to determine the mechanism of AO85-induced exon 45 skipping.

Results

Cell-free splicing of pre-mRNA

Synthesized pre-mRNA was incubated with HeLa cell nuclear extract and the resulting products were analyzed by RT-PCR of total RNA extracted from the reaction mixture. Amplification of a fragment extending from exons A to B revealed 3 products on agarose gel electrophoresis (Fig. 1b). The largest product corresponded to the substrate pre-mRNA that maintained the 3 exons (exons A, 45, and B) and 2 introns (introns A and 45). The middle-sized product lacked the upstream intron A from the pre-mRNA (Δintron A), indicating a splicing intermediate. The smallest band lacked both introns (introns A and 45), corresponding to the mature mRNA. All 3 products showed the expected exon/exon junction sequences and there were no unexpected splicing products caused by cryptic splice site activation. Mature mRNA was obtained most abundantly at 30°C compared with at 37°C or 40°C (data not shown), as reported previously (Habara et al., 2009).

We then monitored the time-course of the splicing reaction by semiquantitative RT-PCR (Fig. 2a). With an incubation time of zero, only the pre-mRNA was detected. At 15 mins, Δintron A was also detected. This indicates that the 5′ intron, intron A, was spliced first in this cell-free splicing system. At 30 minutes' incubation, the amount of Δintron A increased and the mature mRNA was detected. Trace amounts of Δexon 45 were also detected (Fig. 2a).

FIG. 2.

Time-course of the cell-free splicing reaction. (a) Capillary gel electrophoretic patterns of RT-PCR products. At 15 minutes, Δintron A only was visible. From 30 minutes onwards, 3 spliced products were detected, as indicated schematically on the right. Boxes and bars indicate exons and introns, respectively. The GAPDH RT-PCR products are shown at the bottom. (b) When the 15-minute lag period was taken into consideration (corrected time), the percentage of spliced product fitted a pseudo first-order model, with a rate constant (K) of 4.5 hour^–1. Values represent the means for 3 measurements. GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

We then analyzed the percentage of spliced product. Because the Δintron A splicing product appeared at 15 minutes (Fig. 2a), the first 15 minutes was considered an initial lag period (Hicks et al., 2005). When the 15-min lag period was taken into consideration, the appearance of spliced products followed a profile characteristic of a first order reaction (Fig. 2b). The product appearance data fitted the pseudo-first order rate description Y=C×(1–e^–kt), where Y is the fraction spliced, C is the fraction spliced at the end-point of the reaction, k is the apparent rate constant, and t is the time (Hicks et al., 2005). The observed rate constant was 4.5±0.42 hour^–1, fitting well to the 1-phase association model with R²=0.9905 (Fig. 2b) and confirming that this cell-free splicing reaction is time dependent. In the following experiments, the incubation time was shortened to 30 minutes.

Induction of exon 45 skipping with AO85

To examine whether AO85 was able to induce exon 45 skipping in the cell-free splicing system, 0–160 nM AO85 was added to the incubation mixture, and the resulting RNAs were analyzed by RT-PCR. In the absence of AO85, 3 products, corresponding to pre-mRNA, Δintron A, and mature mRNA, were clearly observed (Fig. 3a). At 20 nM AO85, the percentage of Δexon 45 increased, whereas both Δintron A and the mature mRNA decreased. At 160 nM AO85, almost 80% of the total product was Δexon 45. When the percentage of Δexon 45 was plotted against AO85 concentration, the percentage increased linearly (Fig. 3b). The data fitted well to a dose–response model, with R²=0.9990. From this, the EC₅₀ was calculated as 58.0 nM.

FIG. 3.

Dose dependency of exon 45 skipping. (a) Capillary gel electrophoretic patterns of RT-PCR products. The indicated concentration (0–160 nM) of AO85 was added to the in vitro splicing system. The intensity of Δexon 45 increased with increasing concentration of AO85, in contrast to the intensity of the normally spliced band, as indicated schematically on the right. Boxes and bars indicate exons and introns, respectively. The GAPDH RT-PCR products are shown at the bottom. (b) The data fitted well to a linear dose–response model. The EC50 was calculated as 57.96 nM. Values represent the means for 3 measurements. AO, antisense oligonucleotide; EC50, half-maximal effective concentration.

Competitive fashion of AO85 action

We next analyzed action patterns of AO85 in a total splicing reaction by measuring the production of mature mRNA with different concentrations of AO85 and pre-mRNA. The amount of pre-mRNA ranged from 0 to 10.5 nM with 3 different AO85 concentrations (0, 20, and 40 nM). The amount of mature mRNA was normalized to that of GAPDH. On a Lineweaver-Burk plot (Fig. 4a), the data showed that AO85 inhibited the production of mature mRNA in a dose-dependent manner and that 3 lines linking spots met together at the Y axis, indicating a competitive fashion of AO85 action. The Ki of the inhibition was found 10.1 nM (Fig. 4b).

FIG. 4.

Competitive fashion of of AO85 action. (a) Lineweaver-Burk plot for the production of mature mRNA for 3 concentrations of AO85 and various concentration of pre-mRNA. The ratio of normally spliced mRNA to GAPDH was plotted against 1/pre-mRNA concentration (0–10.5 nM). The concentrations of AO85 were 0, 20, or 40 nM (circles, squares, and triangles, respectively). The results showed competitive inhibition of AO85. Values represent the means of 3 experiments. (b) Nonlinear regression of production of mature mRNA in 3 concentration of AO85 and various concentration of pre-mRNA. The ratio of normally spliced mRNA was plotted against the substrate pre-mRNA concentration (0–10.5 nM). The concentration of AO85 was 0, 20, and 40 nM (circle, box, and triangles, respectively). The Ki value was calculated as 10.1 nM. Values represent means from 3 experiments.

Discussion

We showed that AO85 induced exon 45 skipping in a cell-free splicing system that contained substrate pre-mRNA and HeLa cell nuclear extract, indicating that exon skipping does not require intact cells. Regardless of the shortened introns (Fig. 1) the pre-mRNA was subjected to exon skipping. The in vitro splicing assay has been shown useful to examine antisense-mediated exon skipping, although there are limitations in applying the results in understanding in vivo splicing (Spitali et al., 2009). One of the most important limitations is that in vitro splicing requires the use of relatively short transcripts (approximately 1,000–2,000 bp) that usually lack the complexity and length of natural pre-mRNA, and thus may not identify the rate limiting steps for expression in vivo (Nasim et al., 2002). In our in vitro splicing system total amount of RNA decreased at the high concentration of AO85 (Fig. 3a). This may be due to tight connection of AO85 to the target sequence, hampering PCR amplification or unknown RNAse activation.

The cell-free splicing reaction had an initial lag period of up to 15 minutes (Fig. 2). Regulatory elements located within introns and exons guide the splicing complex, the spliceosome, and auxiliary RNA-binding proteins to the correct sites for intron removal and exon joining (Pandya-Jones and Black, 2009; Ward and Cooper, 2010). Presumably, exogenously added pre-mRNAs are initially coated with heteronuclear ribonucleoproteins (hnRNPs) that are abundant in nuclear extracts, and the competing association and dissociation between hnRNPs and components of the spliceosome requires time (Hicks et al., 2005). We assume that the observed lag period corresponds to the spliceosome association. After this initial phase, the splicing reaction proceeded linearly.

AO85, which was identified through trial-and-error, is considered to bind to an exon splicing enhancer (Takeshima et al., 2011). Nuclear proteins bound to the enhancer sequence can promote exon definition by directly recruiting other splicing factors and/or by antagonizing the action of nearby silencer elements. Thus, failure of nuclear proteins to bind to the enhancer sequence would prohibit recognition of the exon by the splicing machinery and cause exon skipping (Takeshima et al., 1995; Pramono et al., 1996; Freier and Altmann, 1997; Stahel and Zangemeister-Wittke, 2003; Aartsma-Rus et al., 2009b; Popplewell et al., 2009). AO85 is expected to bind to a region targeted by 2 SR proteins, SRp30c and SRp40 (Mathews et al., 1999), suggesting that AO85 competes with these 2 SR proteins for binding the pre-mRNA. The binding site for SRp30c is partially located where the pre-mRNA adopts an open secondary structure, whereas the predicted binding site of SRp40 is located within a closed structure (Mathews et al., 1999; ZUKER, 2003). AO85 probably competes with SRp30c, but this needs further examination. Our results showing a competitive fashion of AO85 action (Fig. 4a) support the idea of competition with nuclear proteins.

Our study of the kinetics of AO85-mediated exon skipping in in vitro splicing revealed that (1) AO85 induced exon 45 skipping by inhibiting the production of mature mRNA, with a Ki of 10.1 nM. This relatively low Ki value indicates that AO85 is a potent competitor in the recognition of exon 45, and (2) the EC₅₀ of AO85 was 58 nM. The IC₅₀ of AOs in gene knockdown has been determined to be between 70 and 220 nM (Grunweller et al., 2003), indicating that AO85 works efficiently in our in vitro splicing system. At 160 nM AO85, exon skipping was induced in approximately 80% of transcripts. In comparison with this, at most 70% of exon skipping has been induced by 500 nM 2′-O-methyl phosphorothioate AOs in another cell-free splicing reaction (Spitali et al., 2009). The recent efforts to improve exon skipping efficiency by optimizing the AO sequence, backbone chemistry, and additional modifications (Kurreck et al., 2002) are worthwhile. Considering that AO85 has low EC₅₀ (58.0 nM) (Fig. 3b), high exon skipping efficiency (80%) (Fig. 3b), and ability to induce exon 45 skipping in cultured human muscle cells (Takagi et al., 2004b), we believe that AO85 has high potential for clinical use. However, further studies are necessary before clinical use.

Footnotes

Acknowledgments

We would like to thank Ms. Kanako Yokoyama for her administrative assistance. This work was supported by a grant from the New Energy and Industrial Technology Development Organization, a Grant-in-Aid for Scientific Research (B) and a Grant-in-Aid for Exploratory Research from the Japan Society for the Promotion of Science, a Health and Labour Sciences Research Grant for Research on Psychiatric and Neurological Diseases and Mental Health, and a research grant for Nervous and Mental Disorders from the Ministry of Health, Labour, and Welfare, Japan.

Author Disclosure Statement

No competing financial interests exist.

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