Morpholino-induced exon skipping stimulates cell-mediated and humoral responses to dystrophin in mdx mice

Abstract

Exon skipping is a promising genetic therapeutic strategy for restoring dystrophin expression in the treatment of Duchenne muscular dystrophy (DMD). The potential for newly synthesized dystrophin to trigger an immune response in DMD patients, however, is not well established. We have evaluated the effect of chronic morpholino (PMO) treatment on skeletal muscle pathology and asked whether sustained dystrophin expression elicits a dystrophin-specific autoimmune response. Here, two independent cohorts of dystrophic mdx mice were treated chronically with either 800 mg/kg/month PMO for 6 months (n=8) or 100 mg/kg/week PMO for 12 weeks (n=11). We found that significant muscle inflammation persisted after exon skipping in skeletal muscle. Evaluation of humoral responses showed serum-circulating antibodies directed against de novo dystrophin in a subset of mice, as assessed both by Western blotting and immunofluorescent staining; however, no dystrophin-specific antibodies were observed in the control saline-treated mdx cohorts (n=8) or in aged (12-month-old) mdx mice with expanded “revertant” dystrophin-expressing fibers. Reactive antibodies recognized both full-length and truncated, exon-skipped dystrophin isoforms in mouse skeletal muscle. We found more antigen-specific T-cell cytokine responses (e.g., IFN-g, IL-2) in dystrophin antibody-positive mice than in dystrophin antibody-negative mice. We also found expression of major histocompatibility complex class I on some of the dystrophin-expressing fibers along with CD8+ and perforin-positive T cells in the vicinity, suggesting an activation of cell-mediated damage had occurred in the muscle. Evaluation of complement membrane attack complex (MAC) deposition on the muscle fibers further revealed lower MAC deposition on muscle fibers of dystrophin antibody-negative mice than on those of dystrophin antibody-positive mice. Our results indicate that de novo dystrophin expression after exon skipping can trigger both cell-mediated and humoral immune responses in mdx mice. Our data highlight the need to further investigate the autoimmune response and its long-term consequences after exon-skipping therapy.

Introduction

Duchenne muscular dystrophy (DMD) and its milder allelic form, Becker muscular dystrophy (BMD), are X-linked neuromuscular disorders caused by mutations in the dystrophin gene [1]. The main difference between the two diseases is the severity of the mutation affecting the degree of dystrophin expression, which is largely determined by the reading frame of the spliced mRNA transcript. In DMD, the mutations result in out-of-frame transcripts, leading to an absence of functional dystrophin protein at the myofiber plasma membrane; in BMD, in-frame deletions maintain the transcript reading frame, leading to the production of a truncated and partially functional dystrophin isoform [2].

DMD pathogenesis is characterized by progressive weakness of skeletal, cardiac, and respiratory muscles [3]. A common treatment option for individuals diagnosed with DMD includes palliative treatment with corticosteroids, whose long-term administration is associated with potential side effects [4, 5]. Recent molecular approaches have been found to enable the efficient expression of truncated, BMD-like dystrophin protein without evident adverse effects [6]. Currently, the most promising therapeutic strategy is exon skipping using antisense oligonucleotides (AO), in which the drugs alter mRNA splicing, converting out-of-frame, loss-of-function transcripts into in-frame, functional mRNA transcripts [7–14]. Human exon-skipping clinical trials in DMD patients have resulted in sporadic, localized dystrophin restoration that has been compounded by high variability in terms of both exon-skipping efficiency and observed clinical benefit [9, 15].

In the context of gene therapy, concerns have been raised that synthesis of de novo dystrophin protein might initiate an immunological response in an otherwise dystrophin-naïve DMD patient. Such an immune response has been reported after intramuscular administration to DMD patients of recombinant adeno-associated viral vectors (rAAV) containing a functional mini-dystrophin transgene, with this treatment initiating a strong T-cell-mediated response to both the vector and dystrophin [16, 17]. However, similar concerns were not raised for the AO-mediated exon-skipping approach using the phosphorodiamidate morpholino oligomer (PMO), likely because morpholinos are uncharged and are therefore not expected to elicit an immune response by themselves [18]. Nevertheless, identification of dystrophin-specific T-cell responses to the transgene after gene therapy suggests that sustained dystrophin expression in skeletal muscle could potentially elicit an immune response to restored dystrophin [17, 19]. In the present study, we show evidence that sustained dystrophin expression induced by chronic PMO treatment in mdx mice can induce T-cell and humoral responses to newly synthesized truncated dystrophin protein in some mice. The significance of the anti-dystrophin immune response for the long-term success of exon skipping therapy requires further investigation.

Materials and methods

Animal preclinical studies

All mouse experiments were conducted in accordance with the Institutional Animal Care and Use Committee (IACUC) guidelines at Children’s National Health System and the National Institutes of Health. Five-week-old male mdx (C57BL/10ScSn-mdx/J) (n=16) and wild-type C57BL/10 (n=5) mice were purchased from the Jackson Laboratory (Bar Harbor, ME, USA). All mouse strains were housed at the Children’s National Research Animal Facility under 12-hour light/dark cycles; standard mouse chow and water were provided ad libitum. Dog tissue samples were provided by Dr. Peter Nghiem (Texas A&M University) and obtained in accordance with their institution’s IACUC guidelines. Human biopsies are deidentified and were obtained by Dr. Eric Hoffman and granted an IRB exemption for use in this study by the Children’s National Institutional Review Board (IRB).

Administration of the phosphorodiamidate morpholino oligomer (PMO)

Mice were anesthetized using 4% isofluorane and 0.5 L/min 100% oxygen and then maintained using 2% isofluorane and 0.5 L/min oxygen delivered via a nose cone with a passive exhaust system on a warming device 37). The PMO targeting exon 23 (+07–18; 5’-GGCCAAACCTCGGCTTACCTGAAAT-3’) against the boundary sequences of exon 23 and intron 23 of the mouse dystrophin gene was synthesized by Gene Tools (Philomath, OR, USA). The morpholino antisense drug was administered via systemic delivery through the retro-orbital sinus, as previously described [20, 42], either at monthly intervals for 6 months (800 mg/kg, monthly) or at weekly intervals for 12 weeks (100 mg/kg, weekly). Control mdx mice were injected with saline, and wild-type C57BL/10 mice were used as dystrophin-sufficient controls.

Tissue and serum collection

Mice were sacrificed one month after the final PMO dose. Mice were euthanized using carbon dioxide inhalation, and muscles, organs, and blood were harvested. Blood was collected directly from the heart by cardiac puncture and subsequently centrifuged to isolate the serum. Serum was aliquoted prior to freezing. Tissues (muscles and organs) were surgically removed at the time of necropsy and flash-frozen in liquid nitrogen-chilled isopentane. All serum and tissue samples were stored at −80°C for analysis.

Western immunoblotting

Protein was extracted from mdx-23 and C57BL/10 frozen muscle using RIPA buffer (50 mM Tris-HCl, pH 8.0, with 150 mM sodium chloride, 1.0% Igepal CA-630 [Nonidet P-40], 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate) (Sigma Aldrich, St. Louis, MO, USA; Thermo Fisher Scientific, Waltham, MA, USA) containing protease inhibitors (Halt protease inhibitor mixture 100X, Thermo Fisher Scientific). Extracted protein from mdx-23 and C57BL/10 muscle was loaded in all wells and separated on a Tris-acetate 3–8% gel (Life Technologies, Carlsbad, CA, USA), then transferred overnight at 4 °C to 0.45μm nitrocellulose membranes (Life Technologies). Membranes were stained with Ponceau S (Life Technologies); lanes were cut at MW 238 kDa in order to retain the dystrophin region (460–238 kDa). Membranes were blocked using 5% milk in TBS-Tween (0.1% Tween-20; Sigma Aldrich) and incubated overnight at 4 °C with sera from treated mice at a 1:50 dilution (PMO monthly trial) or 1:150 dilution (PMO weekly trial) or with Dys1 monoclonal antibody (1:1000; Leica Microsystems, Wetzlar, Germany) as a positive control. Membranes were then washed and probed with polyclonal rabbit anti-mouse HRP antibody (1:3000; DAKO/Agilent, Santa Clara, CA, USA) for 1 h at room temperature. Next, the membranes were incubated with ECL WB Substrate (GE Healthcare, Chicago, IL, USA) and developed using the ChemiDoc Touch Imaging System (Biorad, Hercules, CA, USA).

Immunofluorescence staining

Frozen muscle samples from normal mouse, dog, and human biopsies were sectioned at 7-μm thickness and stored at −80 °C until use. Muscle sections were brought to room temperature and fixed with 100% acetone. After washing, the sections were blocked with 10% goat and horse serum (Genetex, Irvine, CA, USA), followed by incubation overnight at 4 °C in a humidified chamber with anti-dystrophin (1:150 dilution; Genetex, GTX15277) as a positive control and with mouse serum at a 1:2 (PMO monthly trial) or 1:5 (PMO weekly trial) dilution. Next, the sections were washed and probed with goat anti-rabbit IgG-Alexa antibody or rabbit anti-mouse IgG-Alexa antibody (1:500; Life Technologies) at room temperature for 1 h and counter-stained with 4’,6-diamidino-2-phenylindole (DAPI)-containing Prolong Gold Antifade agent (Life Technologies) for nuclear staining. Similarly, for staining of the complement membrane attack complex (C5b-9), mouse muscle sections were brought to room temperature and fixed with 4% paraformaldehyde for 10 min. After washing, the sections were permeabilized with PBS-Triton for 10 min. Sections were then washed and blocked in 10% normal donkey serum (Genetex) for 1 h at room temperature, followed by overnight incubation in a humidified chamber at 4°C with rabbit polyclonal anti-C5b-9 antibody (1:250 dilution; Abcam, ab55811, Cambridge, United Kingdom). Next, after washing, the sections were incubated with donkey anti-rabbit IgG-Alexa antibody (H+L) (1:500; Thermo Fisher Scientific, A-21207) for 1 h and counterstained with 4’,6-diamidino-2-phenylindole (DAPI) for nuclear staining. The stained tissue sections were stored at 4°C for imaging and quantification analyses. Microscopy was performed using an Olympus BX61 VS120-S5 Virtual Slide Scanning System with a UPlanSApo 40x/0.95 objective, Olympus XM10 monochrome camera, and Olympus VS-ASW FL 2.7 imaging software (Olympus, Tokyo, Japan). Analysis and quantification were performed using CellSens 1.13 (Olympus), MetaMorph Premier 7.7.0.0 (Molecular Devices, San Jose, CA, USA), Adobe Photoshop CS6 (Adobe, San Jose, CA, USA), and ImageJ (NIH, Bethesda, MD, USA) software.

T-cell responses to dystrophin

Lymph node cells (5.5×10⁵/well) were stimulated with peptides ((Peptide 1: KRLDVDITELHSWIT; Peptide 2: EERLGKLQNHIKTLQ; Peptide 3: AEILKKQLKQCRLLV) as well as scrambled control peptide (Peptide 4: GHLKIENQELQKRLT) at a final concentration of 10 μg/ml. Culture supernatants were collected, and assays for the various cytokines (IFN-γ, IL-2, IL-4, IL-6, TNF-α, IL-10, IL-13, IL-15, IL-22, IL-17E/IL-25, and IL-17A) were performed using U-PLEX MSD custom panels (MSD, Rockville, MD, USA). The data shown here were collected using the manufacturer’s protocol, but with a 1:4 dilution of the culture supernatants in RPMI. The broad dynamic range of the MSD assays enabled the quantitation of analytes that differed in concentration up to 10,000-fold without additional sample dilution or retesting.

Quantification of myofiber necrosis

Myofiber necrosis was quantified from whole cross sections of the tibialis anterior muscle by immunostaining for membrane attack complex (MAC) and H&E. The average number of necrotic fibers per total area from at least three cross-sections were used for quantification. Fibers were counted as necrotic following MAC immunostaining if they met the following criteria: 1) MAC positive staining and 2) DAPI counterstaining demonstrating mononuclear cell infiltration. For H&E, fibers were counted as necrotic if they met the following criteria: 1) reduced eosin staining and myofibrillar striations, and 2) hematoxylin counterstaining demonstrating mononuclear cell infiltration.

Statistical Analysis

Statistical analyses were performed using unpaired t-test, Mann–Whitney test, or one-way ANOVA where appropriate. Calculations were performed using GraphPad Prism version 6 (GraphPad Software Inc., San Diego, CA, USA), and p<0.05 was considered significant.

Results

Significant inflammation in skeletal muscle persists after gene correction by exon skipping

Dystrophin rescue occurred in all PMO-treated mice (supplementary material, Figure S1A). However, the extent of the dystrophin expression varied among the individual mice and between the two muscle groups analyzed. No dystrophin protein was detected in the skeletal muscle of saline-treated control mdx mice (supplementary material, Figure S1A). Despite de novo expression of dystrophin protein in triceps muscle (supplementary material, Figure S1A), significant inflammatory (F4/80+ macrophage) activity in PMO-treated muscle remained. In some areas, the extent of macrophage infiltration was similar to that in saline-treated mdx muscle (Figure 1A,C), suggesting ongoing muscle damage despite gene correction. Macrophage infiltration was less prominent in some areas with dystrophin-positive fibers, but was prevalent in areas devoid of de novo dystrophin expression, reflecting the role of macrophages in the removal of cellular debris (Figure 1A,B).

Figure 1. Muscle inflammation persists despite dystrophin expression after exon skipping.

(A, B) Immunofluorescence co-labeling of dystrophin (DYS; red) and macrophage marker F4/80 (green), after 6 months of high-dose PMO, indicates inflammation was less prominent in areas with dystrophin-expressing fibers. Magnified inset shown in panel B (as defined in panel A). (C) Saline-treated control triceps stained for dystrophin and F4/80 showing substantial macrophage infiltration. Scale bars 100 μm.

Exon skipping can induce the generation of antibodies against dystrophin

To understand the long-term immune consequences of dystrophin expression, we evaluated the effect of chronic PMO treatment on skeletal muscle pathology and asked whether sustained dystrophin expression elicits a dystrophin-specific autoimmune response. Exon-skipping efficiency and dystrophin restoration were assessed after chronic PMO administration, in which mdx mice received high-dose intravenous delivery of 800 mg/kg PMO at monthly intervals for 6 months. PMO-induced dystrophin protein expression was verified by Western blotting (WB) in two typically high-responding muscle groups: triceps and gastrocnemius [20]. Dystrophin rescue occurred in all PMO-treated mice, even though the extent of the dystrophin expression varied widely among the individual mice and between the two muscle groups [20]. No dystrophin protein was detected in the saline-treated control mdx mice (Table 1, usupplementary material, Figure S1A).

Table 1.

Dystrophin restoration after exon skipping, and antibody-response status.

Cohort	ID	Dystrophin Expression	Antibody Status by WB	Antibody Status by Immunofluorescence
Cohort 1 800mg/kg, once a month for 6 months by IV route	PMO-1	Yes	Yes	Not done
	PMO-2	Yes	No	Not done
	PMO-3	Yes	No	Not done
	PMO-4	Yes	No	No
	PMO-5	Yes	No	Not done
	PMO-6	Yes	Yes	Not done
	PMO-7	Yes	Yes	Yes
	PMO-8	Yes	No	No
	CTL-1	No	No	No
	CTL-2	No	No	No
	CTL-3	No	No	No
	CTL-4	No	No	No
	CTL-5	No	No	No
	CTL-6	No	No	No
	CTL-7	No	No	No
	CTL-8	No	No	No
Cohort 2 100mg/kg, once a week for 3 months by IV route	PMO-A	Yes	No	No
	PMO-B	Yes	No	No
	PMO-C	Yes	Yes	Yes
	PMO-D	Yes	No	No
	PMO-E	Yes	Yes	Yes
	PMO-F	Yes	Yes	Yes
	PMO-G	Yes	No	No
	PMO-H	Not done	Yes	Yes
	PMO-I	Yes	Yes	Yes
	PMO-J	Yes	No	No
	PMO-K	Yes	Yes	Yes
	CTL-A	No	No	No
	CTL-B	No	No	No
	CTL-C	No	No	No

^*Percent dystrophin expression relative to wild-type reported for triceps. Bold denotes PMO-treated mice that generated anti-dystrophin antibodies; “Not done” indicates insufficient muscle or serum sample. CTL refers to saline-treated control mice.

To assess whether newly synthesized dystrophin could elicit an immune response and, as a result, lead to the production of dystrophin-specific antibodies, we tested terminal serum collected after termination of PMO and saline treatment (cohort 1). The presence of circulating antibodies to dystrophin was assessed by WB of serum against wild-type mouse skeletal muscle. As a positive control (PC) for dystrophin protein size, one lane was incubated with rabbit polyclonal antibodies against the C-terminus and rod domain of the dystrophin protein (Figure 2A). We observed dystrophin-reactive antibody in the serum (1:50 dilution) from three of the eight PMO-treated mdx mice (37.5% of cohort 1: the mice designated PMO-1, PMO-6, and PMO-7; Figure 2A,C). All three mice with dystrophin-reactive antibody also showed some lower bands whose identity is uncertain at this stage (Figure 2A). The sera from age-matched saline-treated control mdx (n=8) mice showed no reactivity against wild-type dystrophin, indicating an absence of dystrophin-reactive antibody (Figure 2B); however, sporadic non-specific bands were observed at various molecular weights, probably because of the use of unpurified serum as the primary antibody in WB (Figure 2B, black arrows).

Figure 2. Dystrophin-reactive antibodies generated after PMO-induced exon skipping and dystrophin restoration.

(A) Protein extract from dystrophin-sufficient heart muscle was incubated with serum collected from eight mdx mice treated with six monthly doses of 800 mg/kg PMO. Wild-type protein was incubated with Dys1 as a positive control. Three of the eight PMO-treated mdx mice showed serum reactivity against dystrophin: PMO-1, PMO-6, and PMO-7. (B) Serum from saline-treated control mdx mice showed no reactivity against dystrophin. (C) Quantification of relative intensities of dystrophin specificity for Western blots shown in panels A and B. (D) Representative whole-blot imaging shows the specificity of PMO-7 serum for wild-type and PMO-induced “skipped” dystrophin protein; no reactivity was observed in the dystrophin-deficient mdx muscle extract. PC: positive control; SKP: PMO-induced “skipped” dystrophin; WT: wild-type dystrophin. (E–G) Serial wild-type skeletal muscle sections incubated with rabbit polyclonal antibody against dystrophin (E), serum from a PMO-treated mouse (PMO-7) showing reactivity against dystrophin by immunoblotting (F), and serum from a PMO-treated mouse (PMO-4) showing no reactivity against dystrophin by immunoblotting (G). White squares and asterisks indicate continuous muscle fibers between adjacent sections. H-I) Serial sections of wild-type dog skeletal muscle were incubated with control dystrophin antibody (H) or serum from mouse PMO-7 (I). J-K) Serial human skeletal muscle sections were incubated with control dystrophin antibody (J) or serum from mouse PMO-7 (K). Magnified inlays depict the expected dystrophin staining pattern. Scale bars 100 µm. White arrows depict myonuclear staining.

Next, we investigated the specificity of the three dystrophin-positive mouse sera for full-length versus “skipped” dystrophin protein: The results for PMO-7 are shown in Figure 2D and for PMO-1 and PMO-6 in supplementary material, Figure S2B. The positive sera from PMO-7 and PMO-1 recognized dystrophin protein in the form of both full-length wild-type (WT) and truncated dystrophin (Skipped, SKP) (Figure 2D, lane 3 [WT] and lane 4 [SKP], supplementary material, Figure S2B, lanes 3 [WT] and lane 4 [SKP]), whereas PMO-6 recognized only WT full-length, and not skipped dystrophin (supplementary material, Figure S2B, lanes 6 [WT] and lane 7 [SKP]).

Furthermore, PMO-treated sera (PMO-2 to PMO-5) that had previously failed to detect wild-type dystrophin also failed to detect the skipped form of the dystrophin protein (supplementary material, Figure S2D). Overall, these results suggest that the dystrophin-specific positive bands found by WB with unpurified serum against wild-type and PMO-treated mdx muscle extracts were produced by reactive antibodies in response to de novo dystrophin production and are therefore not artifacts reflecting non-specific binding to proteins migrating in the same vicinity as dystrophin.

Dystrophin-deficient muscles often include sporadic clusters of dystrophin-expressing revertant fibers that clonally expand in size with increasing age [21]. To test whether persistent expression of revertant fibers leads to the production of reactive antibodies, we tested serum from two different strains of mdx mice, one on the BL/10 background (mdx-23, n=4), and the other on the BL/6 background (mdx-52, n=4), and their respective wild-type strains, C57BL/10 and C57BL/6 (n=7), at 12 months of age. All serum samples tested were negative for the presence of dystrophin-reactive antibodies (supplementary material, Figure S2A,C). As additional controls, we tested pooled wild-type serum from 8-week-old (n=5) and 7-month-old C57BL/10 mice (n=5), all of which showed no reactivity against the dystrophin protein (supplementary material, Figure S2E), again suggesting that the generation of dystrophin-reactive antibodies in PMO-treated mice was triggered in response to de novo dystrophin production by exon skipping.

Anti-dystrophin antibodies generated after exon skipping recognize sarcolemmal dystrophin in mouse, dog, and human muscle tissue

To confirm that the antibody reactivity found by WB was indeed directed against dystrophin localized to the myofiber sarcolemma, we performed immunostaining of wild-type tissues from three different species using dystrophin-reactive serum. Muscle sections from wild-type dystrophin-sufficient mouse, dog, and healthy human biopsies were individually incubated with serum from PMO-treated mdx mice. First, serial sections from wild-type mouse triceps muscle were incubated with serum from PMO-treated mice PMO-7 (positive reactivity to wild-type dystrophin by WB) (Figure 2F) and PMO-4 (no reactivity to wild-type dystrophin by WB) (Figure 2G). As a positive control, muscle sections were incubated with rabbit polyclonal antibody against the C-terminus of the dystrophin protein (Figure 2E). As observed in our positive controls, serum from mouse PMO-7 produced positive dystrophin staining, specific to the sarcolemma, confirming the reactivity previously found by WB (Figure 2F). In contrast, we found no evidence of dystrophin-specific staining with the serum from PMO-4, which showed no reactivity to wild-type dystrophin by WB (Figure 2G). Similar results were observed when we stained skeletal muscle sections from PMO-treated mdx mice, with the sarcolemmal presence of truncated dystrophin being detected by commercial rabbit polyclonal antibody (supplementary material, Figure S3A) as well as by serum from PMO-7 (supplementary material, Figure S3B), but not from PMO-4 (supplementary material, Figure S3C), confirming that these autoantibodies also recognize skipped dystrophin on skeletal muscle fibers. As expected, the sera from saline-treated mice showed no reactivity with or localization to wild-type mouse dystrophin (data not shown).

Since the dystrophin protein sequence is highly conserved among species, we performed additional immunofluorescent staining analysis using serial sections from dystrophin-sufficient dog and human muscles. Positive control tissues (dog and human) were incubated with the rabbit polyclonal antibody against dystrophin described earlier and showed similar staining results (Figure 2H,J). Dog and human muscle sections incubated with PMO-7 serum showed staining patterns consistent with sarcolemmal dystrophin (Figure 2I,K). Confocal imaging confirmed the expected dystrophin localization, showing the clear interstitial gap between the sarcolemma of adjacent myofibers (Figure 2H–K, see magnified inlays). As expected, PMO-4 did not detect dystrophin from either species (data not shown). Interestingly, with PMO-7 serum, we also observed some myonuclear staining in addition to sarcolemmal staining (Figure 2I,K, white arrowheads), suggesting the presence of immune reactivity to nuclear antigens (e.g., DNA, chromatin, nucleoproteins) in addition to dystrophin.

Frequent low-dose morpholino delivery can also elicit dystrophin-specific antibodies in response to exon skipping

To confirm our findings from cohort 1 and to further evaluate whether the morpholino dosing regimen or level of restored dystrophin protein affects the generation of anti-dystrophin antibodies, we investigated whether weekly low-dose PMO delivery would elicit production of dystrophin-reactive autoantibodies in a second independent cohort of dystrophic mdx mice. Here, mdx mice were treated for 12 weeks with systemic doses of 100 mg/kg/week PMO, and muscle samples and sera were collected 2 weeks after the last dose was administered. Most of the PMO-exposed mice, but none of the saline-treated mice, showed dystrophin expression in the triceps and gastrocnemius muscles by WB (supplementary material, Figure S1B and data not shown).

Generation of dystrophin-reactive antibodies was assessed in cohort 2 mice by WB, with anti-dystrophin antibodies being found in the serum (1:150 dilution) of 6 of 11 treated mice, representing 55% of the PMO-treated cohort. Since most dystrophin-reactive serum samples exhibited only faintly visible dystrophin-specific bands by WB (supplementary material, Figure S5A), we confirmed the reactivity with sarcolemmal dystrophin by immunofluorescent staining of wild-type, dystrophin-sufficient dog muscle. Immunofluorescent staining indeed confirmed sarcolemmal recognition of dystrophin in all six serum samples that showed positivity by WB (Figure 3D–I). None of the samples that were non-reactive in WB showed any immunofluorescent staining of wild-type dog muscle sections (Figure 3C and supplementary material, Figure S4A–D). Overall, these findings further confirm that the synthesis of de novo dystrophin protein in dystrophin-naïve muscle after PMO-induced exon skipping is sufficient to elicit the generation of dystrophin-reactive antibodies within 3 months of PMO delivery.

Figure 3. Localization of dystrophin-reactive antibodies, assessed by immunostaining after prolonged low-dose PMO treatment.

(A–C) Wild-type dog skeletal muscle sections were incubated with rabbit polyclonal antibody against dystrophin (PC, positive control) (A), serum from a saline-treated mouse (CTL-A) (B), or serum from a PMO-treated, dystrophin antibody-negative mouse (PMO-G) (C), with both CTL-A and PMO-G showing no reactivity against dystrophin by WB. (D–I) Wild-type dog skeletal muscle sections were incubated with serum from all mdx mice that showed dystrophin reactivity by WB (PMO-C, PMO-E, PMO-F, PMO-H, PMO-I, and PMO-K). Scale bars represent 100 µm. DAPI counterstaining of nuclei is shown.

T-cell responses to dystrophin peptides are increased in mice with anti-dystrophin antibodies when compared to mice without anti-dystrophin antibodies after exon skipping

We isolated T cells from lymph nodes of mice from the second cohort after exon-skipping treatment. These T cells were stimulated with three dystrophin peptide sequences on either side of exon-23 (Peptide 1 and Peptide 3) and new sequence spanning exon-22–24 (Peptide 2). Culture supernatants were collected, and assays for various cytokines (IFN-γ, IL-2, IL-4, IL-6, TNF-α, IL-15, IL-10, IL-13, IL-15, IL-22, IL-17E/IL-25, and IL-17A) were performed using U-PLEX MSD custom panels. We found consistent increases in antigen-specific IFN-γ, IL-2, and TNF-α levels in the anti-dystrophin antibody-positive group when compared to untreated mdx and the group without anti-dystrophin antibody after exon skipping, suggesting that T-cell activation occurred in the mice with anti-dystrophin antibody (Figure 4). Cytokines IL-4, IL-10, IL-13, IL-15, IL-22, IL-17E/IL-25, and IL-17A were below the detection limit of the assay.

Figure 4. T-cell responses increased in mice with dystrophin-reactive antibodies as compared to mice without dystrophin-reactive antibodies after exon skipping.

Lymph node T cells were stimulated with a cocktail of three dystrophin-specific peptides (100 µg/ml for 72 h), and T-cell cytokines were measured using an MSD custom U-PLEX cytokine panel. (A–C) Cytokine levels, IFN-γ, IL-2, and TNF-α, from PMO-treated and control mdx mice were quantified.

CD8+ cytotoxic T cells can be found in the vicinity of major histocompatibility complex (MHC) class I and dystrophin-positive muscle fibers after exon skipping

To understand the functional consequences of T-cell activation, we evaluated muscle tissues for MHC class I expression and presence of CD8+ cytotoxic T-cells. Generally, MHC class I expression is not present on normal skeletal muscle cells; however, many muscle fibers express MHC class I under autoimmune disease conditions such as myositis [22, 23]. We and others have previously shown that granzyme B from cytotoxic T cells is involved in generating immune responses to self-antigens in skeletal muscle [24]. In the present study, systematic analysis of muscles from PMO-treated mice using the dystrophin rabbit polyclonal antibody and monitoring of T-cell activation demonstrated the presence of lymphocytes around dystrophin and MHC class I double-positive fibers (Figure 5A–C, triangles and arrows); lymphocytes were also present around dystrophin-negative and MHC class I-positive muscle fibers (dots). Not all dystrophin-positive fibers were MHC class I-positive (stars) (Figure 5, upper panel). Co-staining of CD8+ T cells and dystrophin showed several cytotoxic T cells surrounding dystrophin-positive muscle fibers (arrows) (Figure 5D–F). We also verified that T cells around dystrophin-positive muscle fibers were perforin-positive (arrows) (Figure 5G–I) and likely pathogenic. These data suggest that dystrophin and MHC class I double-positive fibers are likely targets of cytotoxic T cell-mediated damage.

Figure 5. Cytotoxic T cells surrounding dystrophin-positive fibers.

(A–C) Representative immunostaining images for dystrophin and MHC class I expression in skeletal muscle of PMO-treated mice. MHC class I⁺/dystrophin⁺ (filled triangles); MHC class I⁺/dystrophin^- (filled dots); and MHC class I^-/dystrophin⁺ (filled star) fibers are shown, as well as, lymphocytes adjacent to MHC class I⁺/dystrophin⁺ and MHC class I⁺/dystrophin^- (white arrows) fibers (C). (D-F) Representative images for dystrophin (DYS) and CD8 immunofluorescent staining of skeletal muscle from PMO-treated mice. Dystrophin+ fibers (D) and their respective CD8 immunostaining (E) indicate that CD8+ cytotoxic T cells can surround dystrophin-expressing muscle fibers (white arrows) (F). (G–I) Representative immunofluorescent images for dystrophin (DYS) and perforin shows that perforin+ cells can be localized around dystrophin-expressing muscle fibers (white arrows). Scale bars 20 µm.

Consequences of anti-dystrophin antibodies and complement activation

We evaluated whether there are any changes in the number of necrotic/degenerating fibers on H&E sections of TA muscle in saline-treated and PMO-treated groups. We found necrotic/degenerating fibers are notably decreased in PMO-treated mice compared to saline-treated mice but did not observe significant differences between dystrophin antibody positive and dystrophin antibody negative groups (supplementary material, Figure S6). Anti-dystrophin antibodies are unlikely to be directly pathogenic to muscle fibers as dystrophin is an intracellular antigen; however, the distribution and clustering of intracellular antigens drastically change during cell death process, and may expose dystrophin for antibody recognition [25]. Complement-mediated muscle damage has been previously reported in dystrophin-deficient muscle [26]. Since the presence of antibodies is essential to classical complement-mediated response, we tested whether the presence of anti-dystrophin antibodies influenced the deposition of the membrane attack complex (MAC) on muscle fibers. We found that PMO-treated mice who were negative for dystrophin antibodies showed less MAC complex deposition than did the PMO-treated antibody-positive group and/or the saline-treated group, although these differences were not found to be statistically significant due in part to our limited sample size (Figure 6A–D). Overall, these findings suggest that muscle fibers in the PMO-treated antibody-negative group are significantly healthier than those of the saline and PMO-treated antibody-positive groups (Figure 6A–D).

Figure 6. Membrane attack complex-positive fibers are decreased in the PMO-treated anti-dystrophin antibody-negative group when compared to the saline-treated control and PMO-treated anti-dystrophin antibody-positive groups.

(A-C) Immunostaining for MAC/C5b-9 (red; left panel), MAC/C5b-9 and DAPI (red and blue, respectively; center panel), and dystrophin (green; right panel) for saline-treated mdx TA muscle (A), PMO-treated, anti-dystrophin antibody positive mdx TA muscle (B), and PMO-treated, anti-dystrophin antibody negative mdx TA muscle (C). Images correspond to serial muscle sections. Scale bars 100 µm. BV – blood vessel. (D) Quantification of MAC/C5b-9 positively-stained fibers for cohorts displayed in A-C.

Discussion

Systemic delivery of AOs is currently the most promising approach for rescuing the expression of a functional dystrophin protein and has recently been approved for use in DMD by the Food and Drug Administration (FDA). At present, there are no reports of dystrophin immunogenicity in the context of exon skipping. In fact, Flanigan et a. found no evidence of dystrophin immunity in any of the 12 patients treated for 6 months with PMO targeting DMD exon 51 in the eteplirsen clinical trial [27]. Unlike mdx mouse studies, in which dystrophin expression is clearly associated with functional improvements, it is challenging to interpret the human eteplirsen clinical trial data because of the small sample size and lack of a correlation between clinical benefit, drug dosage, and dystrophin abundance. It is important to note that in generating an anti-dystrophin immune response, the antigen (dystrophin) needs to be consistently expressed to initiate an immune response. Other approaches for rescuing dystrophin, such as genetic transfer of the dystrophin gene or myoblast stem cell transplantation, have shown the presence of humoral and cellular immune responses against the newly synthesized dystrophin protein in both DMD patients and dystrophic animal models of DMD [16, 17, 27–29]. Here, we found that chronic PMO treatment in two independent cohorts of mdx mice induced truncated dystrophin protein expression in skeletal muscle, and a significant proportion of the PMO-treated mice generated cell-mediated and humoral responses, likely against dystrophin, while none of the saline-treated controls showed any immune response.

We also found that the majority of the anti-dystrophin autoantibodies were reactive with both full-length and skipped mouse dystrophin protein. One of the three mice (PMO-6) in cohort 1 that showed reactivity with full-length mouse dystrophin did not show reactivity with mdx “skipped” dystrophin protein by WB. Possible factors contributing to this absence of dystrophin-specificity for the mdx “skipped” dystrophin protein could be 1) the lower antibody titers in this sample (supplementary material, Figure S2B, lane 6–7) and/or 2) the relatively low amount of restored dystrophin protein in the skipped skeletal muscle.

In our study, mouse anti-dystrophin antibodies recognized dystrophin protein from dog and human muscles as well as mouse muscle, suggesting that immunodominant epitopes are likely conserved in all three species. In fact, the sequences of the longest dystrophin isoforms of humans, dogs, and mice have approximately 92% sequence similarity [30], which would explain the antibody cross-reactivity among species. We also observed mild myonuclear staining in addition to dystrophin-specific staining, hinting at the presence of unknown antibodies that recognize nuclear antigens and indicating the possibility of inter-molecular epitope spreading. Epitope spreading is a phenomenon that refers to the development of an immune response to epitopes distinct from the original immune-reactive epitope [31]. Although we were unable to determine the specific antigens or epitope(s) with which these antibodies reacted, it is plausible that an immune response is mounted to other autoantigens within this milieu [32]. Previous studies have demonstrated the presence of complement activation products, including MAC deposition on muscle fibers in DMD. The source of complement activation in the absence of antibodies is likely the alternative pathway and/or the mannose-binding pathway. Gene correction in the absence of anti-dystrophin antibodies should reduce both the alternative and classical complement activation pathways. We did not find significant differences in MAC deposition between antibody-positive and the saline-treated control group; we found however that the PMO-treated antibody-negative group showed reduced expression of MAC-positive fibers, suggesting that in the absence of antibody responses, complement-mediated damage to “skipped” muscle is less severe. Overall necrosis/degeneration assessed in H&E stained sections of skeletal muscle was decreased in PMO-treated mice compared to saline-treated mice suggesting that gene correction is effective in reducing muscle degeneration. We did not find differences in the level of necrosis/degeneration by H&E partly because necrosis is mediated not only by MAC deposition but also by multiple pathological pathways.

It is important to note that there is extensive muscle necrosis and degeneration in the muscle microenvironment even after exon skipping (Figure 1). Dystrophin is located intracellularly in healthy cells, however location of dystrophin in dying muscle cells is still unknown. In fact, there is extensive literature suggesting that intracellular autoantigens are clustered in two distinct populations of blebs at the surface of apoptotic cells. The population of smaller blebs contains fragmented ribonucleoprotein autoantigens, such as Ro. The larger blebs (apoptotic bodies) contain nucleosomal DNA, Ro, La, and small nuclear ribonucleoproteins, suggesting that both intracellular/cytoplasmic and nuclear antigen localization alter significantly in apoptotic cells [25]. This suggests that there is potential for dystrophin recognition by antibodies during the cell death process and activation of complement pathway accelerates muscle fiber degeneration and necrosis.

We have previously shown that autoantigens are enriched in MHC class I-expressing regenerating muscle cells and are potential targets of cytotoxic T-cell attack in autoimmune myositis [24]. In this study, we have demonstrated the presence of MHC class I- and dystrophin-positive muscle fibers, perforin-positive cytotoxic CD8+ T cells in the vicinity of dystrophin-expressing muscle fibers, and a significant antigen-specific T-cell cytokine response (IFN-γ, IL-2, and TNF-α) in mice positive for anti-dystrophin antibodies. A potential dystrophin-specific CTL response against dystrophin-expressing muscle fibers after exon skipping is likely pathogenic. It has been demonstrated that “blocking” anti-CD8 antibodies can suppress autoreactive cytotoxic CD8+ T-cell activation in a relatively selective manner in mouse models of diabetes [33]. Recent studies have also demonstrated the presence of pre-existing circulating T-cell immunity to dystrophin in a sizable proportion of DMD patients [27]. The induction of T cell cytokine responses against dystrophin peptides in untreated mice indeed indicates some preexisting immunity that is similar to antibody-negative PMO treated mice. Other studies have shown that T cells, especially cytotoxic T cells, are the main mediators of tissue damage in dystrophin-deficient mdx mice [34, 35].

In the context of dystrophin gene transfer with AAV vectors, previous studies in DMD patients have reported immune responses against the vector as well as the dystrophin transgene. One particular study showed that T-cell immunity to dystrophin was present even before gene transfer therapy started [17]. The argument was that the presence of revertant fibers, which arise from various splicing events during RNA processing, could have primed the immune system. Despite this report in DMD patients, we observed no “spontaneous” humoral response to revertants in any of our aged, untreated cohorts, whose ages ranged from 8 to 12 months, a time at which the level of revertants is relatively high in the standard C57/Bl10-mdx mouse model [21]. A key difference between gene transfer and exon skipping is that the AAV vector components interact with the innate immune system. For example, apart from eliciting pro-inflammatory NF-kB activation, the AAV vectors also interact with the innate immune system via the single-stranded DNA genome and Toll-like receptor (TLR) 9/MyD88 and type I interferon pathways [36, 37] and induce capsid-specific T-helper 1 antibody responses [38]. In contrast, we demonstrate here that a cell-mediated and humoral response to dystrophin potentially exists, even in the absence of foreign proteins (i.e., AVV vectors, myoblasts). It is important to note that local inflammatory responses during drug delivery (i.e., at the injection site) were avoided in our study by the use of the retro-orbital intravenous route.

It is intriguing that only a subset of the PMO-treated mice generated an immune response against dystrophin, rather than all the mice. It is known that an autoantigen must fulfill three prerequisites in order to induce an effective autoimmune response: it must have an appropriate context, content, and concentration [39]. The dystrophic skeletal muscle possesses the appropriate context to generate an immune response because it is enriched in pro-inflammatory cells, including myeloid and dendritic cells [31]. In fact, Hartigan-O’Connor et al have shown that dystrophic mouse muscle contains 20 times more macrophages and 7 times more dendritic cells than does healthy muscle [40]. The content, in this scenario the newly synthesized dystrophin, is clearly foreign to both the dystrophin-deficient mdx mice and DMD patients. However, it is important to highlight that gene correction produces variable levels of dystrophin expression, which brings us to the last prerequisite of an effective autoimmune response, concentration. We have recently shown that the amount of dystrophin protein expressed after exon-skipping therapy (concentration) is highly variable among the mice, as well as in different muscle groups within the same mouse [20]. The three prerequisites then suggest that an autoantigen has to be expressed at high enough concentrations and in the appropriate muscle microenvironment in order to elicit an autoimmune response, which was clearly achieved in our study. These criteria could also explain why an antibody response against dystrophin was generated by only a subset, rather than all of the PMO-treated mice in our study.

Although we were unable to assess the impact that reactive antibodies had on the success of exon-skipping therapy, our results show the importance of further investigating immune outcomes in DMD following dystrophin rescue. A reasonable next step would be to investigate whether dystrophin rescue by exon skipping is enhanced in immunodeficient mdx mice or in a multi-modality therapy model with morpholino and immunomodulating drugs.

A model for the anti-dystrophin immune response after gene therapy or exon skipping

Dystrophin-deficient muscle fibers are leaky and release danger-associated molecular patterns (DAMPs). These DAMPs activate innate receptors (Toll-like receptors) on immune cells and muscle triggering the production of pro-inflammatory cytokines. The cytokines then attract additional inflammatory cells into the milieu and produce more cytokines and chemokines that are detrimental to skeletal muscle leading to further muscle degeneration and fibrosis [41].

After exon skipping, muscle fibers expressing dystrophin are less leaky and release lower quantities of DAMPs, and therefore induce less inflammation. However, newly produced dystrophin is processed and presented by antigen-presenting cells (APC) and induces dystrophin-specific CD4 (helper) and CD8 (cytotoxic) T-cell activation and proliferation, leading to anti-dystrophin antibody production and an anti-dystrophin cytotoxic T-cell response. Anti-dystrophin antibody may not be directly pathogenic to muscle but could affect the muscle indirectly through complement activation. However, dystrophin-specific T-cell responses, especially those by CD8+ T cells, may be directly cytotoxic to new dystrophin-positive muscle fibers.

In summary, we have shown in mice that sustained BMD-like dystrophin expression in response to morpholino-induced exon skipping can trigger both cell-mediated and humoral immune responses, and the generation of dystrophin-reactive antibodies may lead to complement activation and muscle cell death, resulting in the variable success of exon-skipping therapies for Duchenne muscular dystrophy.

Supplementary Material

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Supplementary materials and methods NO

Supplementary figure legends NO, because legends are embedded with the figures

Figure S1. Dystrophin expression does not correspond to the generation of dystrophin-reactive antibodies after high-dose or low-dose PMO treatment

Figure S2. Dystrophin-reactive antibodies recognize internally-truncated “skipped” dystrophin protein generated after exon skipping

Figure S3. Dystrophin-reactive antibodies correctly localize to internally-truncated “skipped” dystrophin protein in PMO-treated mdx muscle

Figure S4. Assessment of dystrophin reactivity in PMO- and saline-treated mdx serum

Figure S5. Dystrophin-reactive antibodies are generated after prolonged low-dose PMO treatment