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Keywords:

  • antisense oligonucleotide;
  • dystrophin;
  • Duchenne muscular dystrophy;
  • exon skipping;
  • in vitro screen

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Background

Targeted splice modulation of pre-mRNA transcripts by antisense oligonucleotides (AOs) can correct the function of aberrant disease-related genes. Duchenne muscular dystrophy (DMD) arises as a result of mutations that interrupt the open-reading frame in the DMD gene encoding dystrophin such that dystrophin protein is absent, leading to fatal muscle degeneration. AOs have been shown to correct this dystrophin defect via exon skipping to yield functional dystrophin protein in animal models of DMD and also in DMD patients via intramuscular administration. To advance this therapeutic method requires increased exon skipping efficiency via an optimized AO sequence, backbone chemistry and additional modifications, and the improvement of methods for evaluating AO efficacy.

Methods

In the present study, we establish the conditions for rapid in vitro AO screening in H2K muscle cells, in which we evaluate the exon skipping properties of a number of known and novel AO chemistries [2′-O-methyl, peptide nucleic acid, phosphorodiamidate morpholino (PMO)] and their peptide-conjugated derivatives and correlate their in vitro and in vivo exon skipping activities.

Results

The present study demonstrates that using AO concentrations of 300 nM with analysis at a single time-point of 24 h post-transfection allowed the effective in vitro screening of AO compounds to yield data predictive of in vivo exon skipping efficacy. Peptide-conjugated PMO AOs provided the highest in vitro activity. We also show for the first time that the feasibility of rapid AO screening extends to primary cardiomyocytes.

Conclusions

In vitro screening of different AOs within the same chemical class is a reliable method for predicting the in vivo exon skipping efficiency of AOs for DMD. Copyright © 2010 John Wiley & Sons, Ltd.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Duchenne muscular dystrophy (DMD) arises from mutations in the DMD gene resulting in out-of-frame DMD transcripts and, ultimately, in the absence of functional dystrophin protein leading to fatal muscle degeneration. Modulation of pre-mRNA splicing using short antisense oligonucleotides (AOs) can induce splice correction of aberrant disease-related transcripts to restore their function 1. Such splice correcting AOs have been shown to correct out-of-frame dystrophin transcripts via exon skipping of specific DMD exons, to yield truncated but functional dystrophin protein and, thus, such AOs have therapeutic potential 2–9.

The therapeutic efficacy of splice correcting AOs for DMD was initially demonstrated in vivo by intramuscular injection of 2′-O-methyl phosphorothioate (2′OMePS) AOs in the mdx mouse model that carries a nonsense mutation in exon 23 of the DMD gene 4. Similarly, local injection of 2′OMePS AOs targeting human dystrophin exon 51 was found to be well tolerated and to partially correct dystrophin expression in DMD patients 10. The major current challenge in developing this therapeutic approach is the identification of optimal AO chemistries for effective systemic dystrophin correction, given that DMD is a systemic disease severely affecting all peripheral muscles, including cardiac muscle 11. The systemic splice correcting efficacy of 2′OMePS AOs was found to be relatively poor after intravenous administration in mdx mice, with significant intermuscle variation 5. Neutrally charged phosphorodiamidate morpholino (PMO) AOs were found to provide higher systemic efficacy; however, with a high dose multi-injection protocol and restored dystrophin, protein levels were low and cardiac correction was absent 12. This has prompted the search for improved splice correcting AO chemistries [e.g. neutrally charged peptide nucleic acid (PNA) AOs] 8 and the investigation of novel AO-peptide conjugate chemistries 8, 9, 13–17.

Novel AOs or peptide-conjugated AOs continue to be developed for DMD (e.g. the recent PNA-Pip series) 13, 14 and, therefore, the availability of reliable, rapid in vitro screens to accelerate AO discovery would be highly advantageous. In vitro screens of AO splice correcting activity have been carried out widely using H2K mdx cells 18 and also using DMD patient-derived cells 19–22; however, it has been reported that such in vitro systems work less well for neutrally charged AOs (e.g. PMO or PNA) and their derivatives 20, 23, which can present transfection difficulties, and that the in vitro activity of different AO chemistries seldom correlates well with their in vivo efficacy in mdx mice. Moreover, in vitro AO screens in other affected DMD cell types (e.g. cardiomyocytes) have not been developed but would be a valuable addition to accelerate AO development for target tissues other than skeletal muscle. In the present study, we investigate the development of a rapid, in vitro screening system, and report for the first time the use of optimized in vitro parameters for the screening of a wide range of known and novel AOs for DMD and correlate this directly with their in vivo splice correcting efficacy. We show that the in vitro efficacy obtained using this optimized system allows the screening of different AO chemistries including charged and uncharged AOs, and yields data predictive of in vivo exon skipping activity in the mdx mouse. In addition, the present study demonstrates the validity of direct comparison of the in vitro exon skipping activities of different AOs that derive from same backbone AO chemistry.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Animals

Six- to 8-week-old mdx mice were used in all experiments (three mice in each of the test and control groups) except for mdx mouse cardiomyocyte culture in which neonatal mice were used (20 mice in the group). The experiments were carried out in the Animal Unit (Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK), according to procedures authorized by the UK Home Office. For intramuscular studies, the tibialis anterior (TA) muscle of each experimental mdx mouse was injected with a 40 µl of AO or AO conjugate formulated with saline at a final concentration of 125 µg/ml. Mice were killed by cervical dislocation at the desired time-points, and muscles were snap-frozen in liquid nitrogen-cooled isopentane and stored at −80 °C.

Cell culture and transfection

H2K mdx myoblasts were cultured as previously reported 8. In brief, cells were cultured at 33 °C in 10% CO2 in Dulbecco's modified Eagle's medium (DMEM) supplemented with 20% fetal calf serum, 2% chicken embryo extract (PAA Laboratories Ltd, Yeovil, UK), and 20 units ml−1 γ-interferon (Roche, Herts, UK). Cells were then treated with trypsin and plated at 2 × 104 cells per well in 24-well plates coated with 200 µg/ml gelatine (Sigma, Poole, UK). H2K mdx cells were transfected 24 h after trypsin treatment in a final volume of 0.5 ml of antibiotic- and serum-free Opti-MEM (Life Technologies, Grand Island, NY, USA). Cell transfection with AO or AO conjugates was conducted in the presence of lipofectin (weight ratio 1 : 2 = oligo : lipofectin) (Life Technologies) according to the manufacturer's instructions or in the absence of lipofectin, as noted. After 4 h of incubation, the transfection medium was replaced with DMEM supplemented medium.

Cardiomyocyte isolation and culture

Neonatal mdx mouse cardiomyocytes were isolated and cultured as previously described 24. To reduce fibroblast content, cells were pre-plated in a 60-mm culture dish and incubated for 2 h at 37 °C. The suspension of non-attached cells, predominantly myocytes, was collected and plated at a density of 1 × 105 cells per well in 24-well plates. After 24 h, plates were washed with fresh medium to remove dead cells/weakly attached cells. Cells were cultured for 2 days to obtain continuous monolayers of spontaneously beating cardiomayocytes. Myocytes were tranfected with transfection reagent (RNAiMAX; Invitrogen, Paisley, UK) in accordance with the manufacturer's instructions.

AOs and peptide-AO conjugates

Details of all AO and peptide-AO conjugates studied are shown in Table 1. PNA and peptide-PNA conjugates were synthesized by Panagene (Daejeon, Korea) or in the Gait Laboratory (MRC LMB, Cambridge, UK); PMO and peptide-PMO conjugates were synthesized by AVI Biopharma Inc. (Corvallis, OR, USA) except for Pip2b-PMO in the Gait Laboratory. Different AO lengths and sequence positions with respect to the murine DMD exon 23 boundary region are shown in Table 1.

Table 1. Antisense oligonucleotide nomenclature: chemistries and sequences
ChemistryNameSequence
  • Nomenclature and sequence details of AO and AO-peptide conjugates designed to target the boundary sequences of exon and intron 23 of the dystrophin gene. Peptides are written from N to C orientation using the standard one letter amino acid code, except for X and B, which are un-natural amino acids (X = 6-aminohexanoic acid, B = β-alanine). The sequence of the muscle targeting peptide MSP is ASSLNIA. AOs and AO-peptide conjugates were assessed in:

  • 1

    H2K mdx myoblasts and

  • 2

    mdx cardiomyocytes.

2′-O-methyl phosphorothioate RNA2′OMePS1,25′-ggccaaaccucggcuuaccu-3′
 RNA-TAT15′-YGRKKRRQRRRP-ggccaaaccucggcuuaccu-3′
Peptide nucleic acid (PNA)PNA15(+2–13)15′-aacctcggcttacct-3′
 PNA16(+2–14)15′-aaacctcggcttacct-3′
 PNA17(−2–18)15′-ggccaaacctcggctta-3′
 PNA18(+2–16)15′-ccaaacctcggcttacct-3′
 PNA18(−1–18)15′-ggccaaacctcggcttac-3′
 PNA20(+2–18)15′-ggccaaacctcggcttacct-3′
 Pip2B-PNA201,25′-((RXR)3IHILFQNdRRMKWHKBC)-ggccaaacctcggcttacct-3′
 (RXR)4-PNA2015′-(RXR)4XB-ggccaaacctcggcttacct-3′
 PNA25(+7–18)15′-ggccaaacctcggcttacctgaaat-3′
 Pip2B-PNA2515′-((RXR)3IHILFQNdRRMKWHKBC)-ggccaaacctcggcttacctgaaat-3′
 (RXR)4-PNA2515′-(RXR)4XB-ggccaaacctcggcttacctgaaat-3′
Morpholino phosphorodiamidate oligomer (PMO)PMO15′-ggccaaacctcggcttacctgaaat-3′
 (RXR)41,25′-(RXR)4XB-ggccaaacctcggcttacctgaaat-3′
 Pip2B15′-((RXR)3IHILFQNdRRMKWHKBC)-ggccaaacctcggcttacctgaaat-3′
 B15′-RXRRBRRXRRBRXB-ggccaaacctcggcttacctgaaat-3′
 MSP-B15′-ASSLNIAX-RXRRBRRXRRBRB-ggccaaacctcggcttacctgaaat-3′
 B-MSP15′-RXRRBRRXRRBRB-ASSLNIAX-ggccaaacctcggcttacctgaaat-3′

RNA extraction and nested reverse transcriptase-polymerase chain reaction (RT-PCR) analysis

Total RNA was extracted from transfected cells with Trizol (Invitrogen) and 200 ng of RNA template was used for 20-µl RT-PCR with the OneStep RT-PCR kit (Qiagen, Crawley, UK). The primer sequences for the initial RT-PCR were: Exon20Fo 5′-CAGA ATTCTGCCAATTGCTGAG-3′; Ex26Ro 5′-TTCTTCAGCTTGTGTCATCC-3′ for reverse transcription from mRNA and amplification of cDNA from exons 20–26. The primer sequences for the second round were: Ex20Fi 5′-CCCAGTCTACCACCCTATCAGAGC-3′ and Ex26Ri 5′-CAGCCATCCATTTCTGTAAGG-3′. The cycle conditions were as previously described 8. The products were examined by electrophoresis on a 2% agarose gel.

Immunohistochemistry

Tissue sections of 8 µm were cut from at least two-thirds of the treated TA muscles at 100-µm intervals. The sections were then examined for dystrophin expression with a polyclonal antibody 2166 against the dystrophin carboxyl-terminal region (the antibody was kindly provided by Professor Kay Davies). The maximum number of dystrophin-positive fibres in one section was counted using a Zeiss AxioVision fluorescence microscope (Carl Zeiss, Oberkochen, Germany). Polyclonal antibodies were detected by goat-anti-rabbit IgGs Alexa Fluro 594 (Molecular Probes, Invitrogen).

Protein extraction and western blot

Protein was extracted from differentiated H2K mdx myotubes treated with different AOs or AO conjugates 2 weeks after transfection as described previously 8. Various amounts protein from normal C57BL6 mice as a positive control and corresponding amounts of protein from treated or untreated H2K mdx myotubes were loaded onto sodium dodecyl sulphate polyacrylamide gel (4% stacking, 6% resolving). The protein transfer and blotting was conducted as described previously 8. An α-actinin (monoclonal antibody, 1 : 5000; Sigma) was used as a loading control. The intensity of the bands obtained from treated mdx myotubes was measured by ImageJ software (http://rsb.info.nih.gov/nih-image/); the quantification is based on band intensity and area, and is compared with that from normal TA muscles of C57BL6 mice.

Cell toxicity assay

A modified WST-1 assay, which measures the metabolic activity of viable cells 25, was used to evaluate the toxicity of different AOs and peptide-conjugated AOs in H2K mdx cells at 1, 5 and 10 µM concentrations. Cells were grown in 96-well microplates overnight and treated with different AOs or peptide-AO conjugates and compared with untreated cells as a control for 12 h, and then incubated with WST-1 for approximately 4 h. During this incubation period, viable cells convert WST-1 to a water-soluble formazan dye which is then quantified using an enzyme-linked immunosorbent assay plate reader assay.

Statistical analysis

All data are reported as the mean ± SEM. Statistical differences between treatment groups and control groups were evaluated by SigmaStat (Systat Software Inc., Chicago, IL, USA) and Student's t-test was applied.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

In the present study, we aimed to develop an optimized in vitro system for the rapid screening of known and novel AO compounds. A wide range of different AO chemistries and their derivatives were evaluated in H2K mdx myoblasts (Table 1) and a series of in vitro conditions and parameters were optimized, including the use of transfection reagents, dose-response and time-course studies. In parallel, a direct comparison was conducted between in vitro and in vivo data to validate the applicability of this in vitro system as a screening tool for subsequent in vivo studies. In addition, we have established a cardiomyocyte cell model for the first time for screening AOs for cardiac exon skipping in DMD.

Optimization of AO in vitro screening: dose-response and time-course studies

To define the in vitro parameters, we studied AO concentration dependency and time-course kinetics in detail. We began with known 2′OMePS AOs by analysing the concentration dependency of this AO and its peptide conjugate derivative (RNA-TAT), in which the well-known, arginine-rich cell penetrating peptide (CPP) TAT derived from HIV was directly conjugated to the 2′OMePS AO. Because of the poor ability of these compounds to enter cells, little or no activity was found with either AO at given concentration in the absence of lipofectin (transfection reagent) and better exon skipping efficacy was detected at the RNA level via RT-PCR analysis with the use of lipofectin (see Supporting information, Figure S1a). In subsequent in vitro studies, lipofectin was used for 2′OMePS and RNA-TAT AOs except where otherwise stated. The splice-correcting efficacy of naked 2′OMePS was found to be superior to RNA-TAT over a range of concentrations from 50 nM to 1 µM, with clear statistically significant differences being detectable at the lowest concentration of 50 nM (Figures 1a and 1b).

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Figure 1. In vitro analysis of different AOs and their peptide conjugates in H2K mdx cells. (a) RT-PCR to detect the dystrophin exon skipping products in treated H2K mdx cells with different concentrations of 2′OmePS and its peptide conjugate (RNA-TAT) in the presence of transfection reagent (lipofectin). Unskipped products or those deleted for exon 23 or exons 22 and 23 are as indicated and (+) indicates that lipofectin transfection reagent was used. (b) Quantification of exon 23 skipping products using ImageJ software. The results show significant differences in the percentage of exon 23 skipping between 2′OMePS and RNA-TAT over the range of tested concentrations. (c) RT-PCR results for PNA AO and its peptide conjugates in H2K mdx cells at different concentrations. Full-length and skipped products for exon 23 or exons 22 and 23 are as shown and (–) indicates the absence of lipofectin, whereas (+) refers to the presence of lipofectin. (d) Quantification of the percentage of exon 23 skipping by ImageJ, which shows significant improvement between peptide-PNA conjugates and PNA AO alone for the percentage of exon 23 skipping. A lower level of exon skipping was observed in PNA AO treated H2K mdx cells in all the tested doses. (e) RT-PCR assay for PMO AO and its peptide conjugates in H2K mdx cells. Full-length and skipped products for exon 23 or exons 22 and 23 as shown. (f) Quantification of the percentage of exon skipping by ImageJ. The data show higher activity with peptide-PMO conjugate than PMO AO at the tested concentrations (n = 6 replicates, *p < 0.05)

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In the case of 20-mer PNA AO and its peptide conjugate derivatives (Pip2b-PNA and (RXR)4-PNA), the superior efficacy of the Pip2b conjugate at the RNA level was evident, being statistically significant at the 300 nM concentration level (Figures 1c and 1d) in the absence of lipofectin. No difference was observed in the presence or absence of lipofectin for PNA and peptide-PNA conjugates at the given concentrations (see Supporting information, Figure S1b). For naked PNA AOs, trace amounts of exon skipping could be seen at 500 nM, which became clearly evident at a concentration of 1 µM in the presence of lipofectin. Therefore, lipofectin was only used for subsequent PNA but not for peptide-PNA conjugates in vitro screening.

Naked PMO AO and its peptide-conjugates have potential for systemic dystrophin correction 9, 12, 15, 17, and hence show significant promise as DMD therapeutics. A similar approach was used to determine the effect of lipofectin on the activity of PMO AO and its peptide conjugate (RXR)4-PMO) in H2K mdx cells. Very little activity was found with PMO AO in the presence and absence of lipofectin. The superior splice correcting activity of (RXR)4-PMO could be detected at the lowest 50 nM concentration and was maintained over the full concentration range in the absence of lipofectin (Figures 1e and 1f). The higher activity of peptide-PMO over naked PMO AO was clearly indicated even in the absence of lipofectin at a given concentration (see Supporting information, Figure S1c) and, therefore, all subsequent screens of peptide-PMO conjugates were conducted in the absence of lipofectin. These data suggest that direct comparisons of AO efficacy could be obtained in vitro within a given backbone chemistry class, which are consistent over a wide dose range permitting selection of one single dose for future comparative studies.

We next aimed to understand the exon skipping kinetics to minimize the need for screening at multiple time-points. In all but one case (RNA-TAT), the data obtained at the 24-h time-point were representative of that obtained at the 48- and 72-h time-points regardless of AO chemistry (Figures 2a and 2b), although the data at 12 h post-transfection were more variable. This suggests that screening for dystrophin exon skipping in the H2K mdx myoblasts can be performed reliably at 24 h after AO transfection. We also showed that, at AO and peptide-AO concentrations of up to 10 µM, which are higher concentrations than required for screening in all three major AO classes examined, any complicating effects as a result of cellular toxicity and/or compromised cell viability could be expected to be negligible (Figure 2c), although a reduction in cell viability was found for the Pip2b-PNA AO conjugate.

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Figure 2. Time-course analysis for different AOs and their peptide conjugates in H2K mdx cells. (a) RT-PCR results for different AOs and their peptide conjugates in H2K mdx cells at different time-points from 12–72 h after transfection. Unskipped products or those deleted for exon 23 or exons 22 and 23 are as indicated; (+) indicatess the presence of lipofectin, whereas (−) indicates the absence of lipofectin. (b) Quantification of percentage of exon 23 skipping for different AOs and their peptide conjugates at different time-points tested. The results show that 24 h post-transfection is the peak time for inducing exon skipping with all the tested AO and peptide conjugates except RNA-TAT in H2K mdx cells (n = 6 replicates). (c) A WST-1 cellular toxicity assays for the tested AOs and their peptide conjugates in H2K mdx cells testing an AO concentration of up to 10 µM. The data reveal that no toxicity was observed for all the compounds except Pip2b-PNA, which showed approximately 80% cell death at 5 and 10 µM as measured by the WST-1 assay (n = 3 replicates)

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Direct comparison of in vitro and in vivo exon skipping activities of 2′OMePS AOs and peptide-conjugated derivatives

To validate the reliability of this optimized screen system for predicting in vivo AO activity, we first evaluated the 2′OMePS AO and its peptide derivative RNA-TAT for activity in H2K mdx myoblasts and compared this directly with in vivo splice correcting efficacy in mdx mice. Significantly greater 2′OMePS activity over the peptide-conjugated derivative was shown in an RT-PCR assay in the presence of lipofectin in H2K mdx cells (Figure 3a) at 300 nM concentration and at 24 h post-transfection as optimized above. The increased activity of 2′OMePS over its peptide derivative was replicated after an in vivo screen by direct intramuscular injection into the TA muscle of 6–8-week-old adult mdx mice, where the number of dystrophin positive fibres was up to 120 in TA muscle cross-sections treated with 2′OMePS AO versus 88 with RNA-TAT as detected by immunofluorescent staining (Figures 3b and 3c), demonstrating good correlation with the in vitro RT-PCR data.

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Figure 3. In vitro and in vivo activity comparisons for 2′OMePS AOs and RNA-TAT. (a) RT-PCR result for H2K mdx cells treated with 2′OMePS and RNA-TAT at a concentration of 300 nM at 24 h post-transfection in the presence of lipofectin. Unskipped and skipped products for exon 23 or exons 22 and 23 are as indicated. The quantification of the exon skipping efficiency at the indicated concentration and time-point is shown as indicated (n = 6 replicates). (b) Local restoration of dystrophin expression after intramuscular delivery of 2′OMePS and RNA-TAT. Immunostaining to detect dystrophin expression in TA muscles of mdx mice treated with local intramuscular injection of 5 µg of 2′OMePS and its peptide conjugate compared to untreated mdx mice and normal C57BL6 mice. (scale bar = 200 µm). (c) Quantification of dystrophin-positive fibres in TA muscles treated with 2′OMePS AO and RNA-TAT intramuscularly. Both treatments show a significant increase in the number of dystrophin-positive fibres compared to age-matched untreated mdx controls, whereas 2′OMePS AOs showed slightly higher activity than RNA-TAT (*p < 0.05)

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In vitro versus in vivo activity comparison for novel PNA AOs and peptide-PNA conjugates

Next, we aimed to extend the study to investigate the neutrally charged PNA AO chemistry and various PNA derivatives. On the basis of the above optimized in vitro conditions, we directly compared the activity of PNA AO and peptide-PNA conjugates in H2K mdx cells at 300 nM concentrations, at 24 h post-transfection, and correlated this with in vivo analysis of efficacy. The order of exon skipping efficiency with neutral PNA AO and its peptide derivatives (Pip2b-PNA and (RXR)4-PNA) in the in vitro screen (Figure 4a) was found to correlate highly with in vivo exon skipping efficacy after intramuscular injection in mdx mice (Figure 4b; see also Supporting information, Figure S2). The standard PNA AO length used in all previous studies 8, 13, 14 and as shown in Figures 1b and 1c and 4a and 4b of the present study was 20 mer, corresponding to a previously optimized exon 23 AO sequence. We hypothesized that PNA activity is likely to be length-dependent and that optimal activity might be obtained with longer and/or shorter PNA AOs. We therefore extended this study to screen a panel of novel PNA AOs of varying lengths in the presence of lipofectin (Table 1). In vitro activity in H2K mdx cells as detected by RT-PCR revealed that both longer (25 mer) and shorter (18 mer) PNA AOs had comparable or improved activity (11%) over the standard 20-mer sequence (2%) as shown by the percentage of exon skipping, which correlated highly with in vivo exon skipping activity after intramuscular injection in mdx mice (Figures 4c and 4d; see also Supporting information Figure S3). The order of exon skipping efficiency between PNA20 and PNA25 was validated by systemic intravenous study 26. Thus, a rapid in vitro screen was highly predictive of the most active PNA25 AO for dystrophin splice correction in vivo. Furthermore, exon skipping efficiency of naked PNA25 AOs was found to be superior to peptide conjugated PNA25 AOs (Pip2b-PNA and (RXR)4-PNA) at 300 nM and at the 24 h time-point after transfection in the presence of lipofectin (Figures 4e and 4f), which again correlated closely with the relative in vivo activities of these compounds (Figure 4g; see also Supporting information, Figure S4).

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Figure 4. Comparison of in vitro and in vivo activity for PNA AOs and peptide conjugates. (a) RT-PCR for PNA AO and its peptide conjugates in H2K mdx cell at 300 nM 24 h after transfection in the presence or absence of lipofectin. Full-length and skipped products with exon 23 or exons 22 and 23 are as indicated. Lipofectin was used for PNA AO transfection and not for peptide-PNA conjugates. The quantification for exon 23 skipped products at the tested concentrations and time-points is as indicated. Results show higher activity with peptide-PNA conjugates than PNA AO in H2K mdx cell. (b) Quantification of dystrophin-positive fibres in TA muscles after single intramuscular delivery of 5 µg of PNA and peptide-PNA conjugates in mdx mice. The data reveal a significant increase in the number of dystrophin-positive fibres in Pip2b-PNA compared to PNA AO alone (*p < 0.05). (c) Screening of different lengths of PNA AO in H2K mdx cells. The results show the highest exon skipping activity with PNA25 compared to other shorter PNA AOs in the presence of lipofectin at a concentration of 1 µM 24 h after transfection (n = 6 replicates). (d) Quantification of dystrophin-positive fibres in TA muscles treated with different lengths of PNA AOs after local intramuscular injection of 5 µg of various PNA AOs in mdx mice (*p < 0.05). (e) Test of PNA25 and its peptide conjugates in H2K mdx cell. RT-PCR data show that higher activity was observed with PNA25 in the presence of lipofectin than peptide-PNA25 conjugate at both 300 nM and 500 nM 24 h after transfection. (f) Quantification of exon skipping induced by PNA25 and peptide-PNA25 at 300 nM and 24 h after transfection in H2K mdx cell (n = 6 replicates). (g) In vivo data for the number of dystrophin-positive fibres in TA muscles treated with 5 µg of PNA25 and peptide-PNA25 intramuscularly in mdx mice. Significant differences in the number of dystrophin-positive fibres are observed between PNA25 and peptide-PNA25 conjugates (*p < 0.05)

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In vitro screen identifies PMO derivatives with high in vivo activity

Finally, we applied this optimized in vitro screening assay to the highly promising PMO chemistry and novel peptide-PMO conjugates. Utilizing the above optimized in vitro conditions (300 nM AO concentrations at 24 h post-tranfection), naked PMO AOs were found to yield dystrophin exon skipping up to 0.9% by RT-PCR assay in the presence of lipofectin, markedly less efficient than two peptide conjugated PMO AOs (Pip2b-PMO and (RXR)4-PMO), which showed 19.7% and 6.1% exon skipping, respectively, in the absence of lipofectin (Figure 5a). These findings again correlated closely with the relative in vivo splice correcting activity of these compounds after intramuscular injection in mdx mice (Figure 5b; see also Supporting information, Figure S5). Furthermore, the correlation can be extended to systemic intravenous study with naked PMO and (RXR)4-PMO 4, 9. A series of novel muscle targeting peptide-PMO conjugates were also screened in vitro, centred on the previously described muscle-specific peptide (MSP) 27. Two chimeric CPP-MSP conjugates were studied and compared with a previously described peptide-PMO conjugate B-PMO 9, 15. RT-PCR data from the in vitro screen showed that both B-PMO and B-MSP-PMO AOs were more effective than MSP-B-PMO at 300 nM concentrations 24 h after transfection (Figure 5c). This was highly correlated with the in vivo activity of these compounds both by direct intramuscular injection and by systemic intravenous injection in mdx mice 28. The splice correcting activity of different AO chemistries in H2K mdx cells was confirmed by western blotting (Figure 5d), which showed that up to 100% dystrophin protein was restored by the (RXR)4-PMO conjugate.

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Figure 5. Comparison of in vitro and in vivo activity between PMO and PMO peptide conjugates. (a) RT-PCR results for PMO and its peptide-conjugates at 300 nM and 24 h post-transfection in H2K mdx cell. Full-length and skipped exon 23 or exons 22 and 23 products are as indicated. The percentage of exon 23 skipping in H2K mdx cell was quantified by ImageJ, in which both peptide-PMO treatments showed a significant improvement compared to PMO AO alone (n = 6 replicates). (b) Quantification of dystrophin-positive fibres in TA muscles treated with 5 µg of PMO and peptide-PMO conjugates by intramuscular injection in mdx mice. Data show (RXR)4-PMO has the highest activity compared to PMO and Pip2b-PMO (*p < 0.05). (c) In vitro evaluation of other peptide-PMO conjugates in H2K mdx cell. RT-PCR showed higher activity with B-MSP-PMO than B-PMO and MSP-B-PMO. The percentage of exon skipping with three peptide-PMO conjugates at 300 nM 24 h post-transfection in H2K mdx cell is as indicated (n = 6 replicates). (d) Western blot for the differentiated H2K mdx myotubes treated with 2′Ome RNA, Pip2b-PNA and (RXR)4-PMO, which are AOs with higher activity in each class of chemical backbone. α-actinin was used as a loading control

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In vitro splice correcting activity of AOs in cardiomyocytes

The above data have shown that in vitro screens within a given chemistry class can be reliably carried out at a single concentration and time-point in a mdx myoblast cell to yield data highly predictive of in vivo exon skipping efficacy after intramuscular injection in the mdx mouse model of DMD. Given that DMD is a systemic disease affecting organs other than skeletal muscle (notably also cardiac muscle), we wished to test whether in vitro AO studies and ultimately screens in cardiomyocytes were feasible. We therefore undertook a preliminary study of a small number of oligonucleotides of different AO classes to optimize conditions for in vitro AO screening in primary mdx mouse cardiomyocytes. A number of different tranfection reagents were tested (data not shown) from which RNAiMax was shown to yield optimal results; this was therefore used for all subsequent studies in mdx mouse cardiomyocytes. Exon skipping efficiency was detectable at the RNA level using RT-PCR. Good activity was detected with 2′OMePS, Pip2b-PNA and (RXR)4-PMO compounds (Figure 6), in which (RXR)4-PMO showed the highest activity and correlated well with the systemic intravenous studies even with the different chemistry of AOs 5, 9, 26.

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Figure 6. In vitro evaluation of different AOs in isolated mdx mouse cardiomyocytes. RT-PCR for different AOs in isolated mdx mouse cardimyocytes in the presence of transfection reagent (RNAiMAX). The results showed that the highest exon skipping efficiency was observed with (RXR)4-PMO compared to other AOs. Unskipped and skipped products for exon 23 and exons 22 and 23 are indicated in the image and (+) indicates RNAiMAX was used, whereas (−) refers to the absence of transfection reagent. The percentage of exon skipping in isolated mdx mouse cardiomyocytes was measured using ImageJ (n = 4 replicates)

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

In the present study, we report the detailed in vitro and in vivo analysis of a wide range of known and novel AO chemistries from different chemical classes, and show that, within a given chemical AO class, reliable in vitro screening in the H2K mdx cells at a single time-point and AO concentration can yield data highly predictive of in vivo splice correcting activity in the mdx mouse. In each class (2′OMePS, PNA and PMO), the AO with superior splice correcting activity was successfully identified in vitro and subsequently shown to be active in vivo after intramuscular injections (i.e. the relative splice correcting efficacy of AOs within a given AO class could be reliably identified in the in vitro screen). We also show for the first time that this work can be extended to an analysis in primary cardiomyocytes, although further optimization of this methodology is needed, and it remains to be shown whether such in vitro activity is predictive of in vivo activity in cardiac tissue. Thus, the in vitro system and the conditions reported in the present study will be valuable for the rapid screening of novel splice correcting AOs, including novel AO chemistries 8 and AO-peptide conjugates 13, and should accelerate future therapeutic advances for DMD.

The earliest demonstrations of successful AO-induced exon skipping of the dystrophin were demonstrated in vitro in human lymphoblastoid and mdx mouse muscle cells 18, 21, skipping DMD exons 19 and exons 22–30, respectively. Subsequently, numerous in vitro exon skipping studies have been undertaken demonstrating precise skipping of DMD exon 23 in mdx mouse muscle cells 29 with protein correction in mdx mice in vivo6; successful DMD exon 46 skipping in patient-derived muscle cells 22; further mdx exon 23 2′OMePS sequence refinement and evaluation of AO concentrations 30; evaluation of ‘leashed’ PMO AOs 23; the comparative effects of different 2′OMePS, PMO, PNA and LNA AOs for skipping DMD exon 46 in human muscle cells 2; the influence of AO length on AO splice correcting efficacy 31; optimization and selection of a human DMD exon 51 skipping AO for clinical trial 32; and the exon skipping activity of PNA and PNA-peptide AOs in mdx muscle cells 8, 13, 14. Despite these and other in vitro studies, little agreement exists on the optimal parameters for in vitro AO screening, no studies to date have compared across different AO backbone chemistries and AO-peptide modifications, and little if any data exist on the value of in vitro screens as predictors of in vivo AO activity across a wide range of AO compounds. Given the range of backbone AO chemistries and AO sequence, length, peptide and other modifications currently available, a high-throughput in vitro screening assay that correlates well with in vivo efficacy would be invaluable and accelerate AO discovery and development.

In the present study, we define a single nanomolar concentration and time-point post-transfection for the screening of AO compounds with a range of backbone chemistries and length and peptide modifications in mdx muscle cells in vitro. Although AO concentrations below 300 nM and at time-points beyond 24 h were useful in many cases, selecting a single concentration of 300 nM and a time-point of 24 h permitted effective comparison. By contrast to a number of previous studies 2, 23, in vitro splice correcting activity was demonstrated for unmodified PNA and PMO Aos, as well as for peptide conjugated versions of these compounds. Importantly, use of the optimized in vitro parameters allowed comparative AO activity to be determined by measuring the percentage of skipped DMD transcript at the RNA level. In each AO class, this therefore identified the most effective AO compound with the relative rather than absolute level of exon skipping being the critical read-out.

Given that novel AOs and AO modifications identified in in vitro assays will be subsequently studied for DMD splice correction and functional restoration in vivo in DMD animal models as a prelude to their use in the clinical studies, which are now in progress 10, an important question concerns the predictive value of any of such comparative in vitro screens. In the present study, we report a detailed comparison of in vitro and in vivo AO activity in mdx mice and demonstrate a high degree of correlation of in vitro and in vivo AO activities within a given AO chemical class. The superior activity of 2′OMePS AOs compared to a TAT conjugated AO, of longer 25 mer PNAs compared to shorter PNAs, and of a range of peptide conjugated PMOs versus naked PMO were all confirmed in vivo in mdx mice, and, in the case of the latter, the very high in vitro activity was matched with high efficiency splice correction in vivo. This therefore provides a rapid route for the future screening of novel AO backbone chemistries and chemical derivatives prior to the detailed in vivo study of high activity lead AO compounds.

Finally, we report data extending such an in vitro AO screening assay to cells from other tissues affected in DMD, notably cardiomyocytes and neurones. Moreover, the acknowledged difficulty experienced to date with respect to effecting successful splice correction in these alternative target cells 12 suggests that the availability of in vitro assays might be of value for identifying the factors affecting the splice correction efficiency in these cells. In the present study, we report the establishment of a primary mdx cardiomyocyte in vitro splice correcting assay and show that DMD splice correction is achievable in this primary cell system using a range of AO compounds, which indicated that no intrinsic barrier existed in the cardiomyocytes and that the low exon skipping efficiency in heart might be a result of the inefficienct delivery of these AO compounds.

In summary, we report the detailed characterization of an in vitro AO screen in mdx mouse muscle cells that permits the screening and detailed comparative analysis of a range of AO backbone chemistries and chemical derivatives, and that also shows a high degree of correlation between in vitro and in vivo activity in mdx mouse muscle; within a given chemical AO class, the relative exon skipping activity of different AO compounds in vitro was found to correlate with their relative efficacy in vivo. This, together with the description for the first time of a primary cardiomyocyte assay, will allow a more efficient screening in the future of larger numbers of novel AOs and, in particular, of modified peptide-AO derivatives and will therefore permit higher throughput in vitro assay systems to be established for AO screening, which will accelerate the clinical development of novel AOs for DMD therapy.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

This work was supported by research grants from Action Duchenne UK to M.J.A.W. The authors acknowledge the UK MDEX Consortium for helpful discussions; Professor Kay Davies (Department of Physiology, Anatomy and Genetics, University of Oxford) for providing access to facilities, including the mdx mouse colony. The authors declare that there are no competing financial or other conflicts of interest.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information
FilenameFormatSizeDescription
jgm_1446_sm_suppinfodata.doc7801KSupporting Information
jgm_1446_sm_suppinfo.doc1621KSupporting Information

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