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The Effect of Immunomodulatory Treatments on Anti-Dystrophin Immune Response After AAV Gene Therapy in Dystrophin Deficient mdx Mice

Abstract

Background:

AAV-based gene therapy is an attractive approach to treat Duchenne muscular dystrophy (DMD) patients. Although the long-term consequences of a gene therapy approach for DMD are unknown, there is evidence in both DMD patients and animal models that dystrophin replacement by gene therapy leads to an anti-dystrophin immune response that is likely to limit the long-term use of these therapeutic strategies.

Objective:

Our objective is to test whether the anti-dystrophin immune response is affected by immunomodulatory drugs in mdx mice after rAAV gene therapy.

Methods:

mdx mice were treated with rAAV microdystrophin alone or in combination with immunomodulatory drugs. Dystrophin expression in skeletal muscle was assessed by mass spectrometry. Immune responses were assessed by immunophenotyping, western blot for anti-dystrophin antibodies and flow cytometry assays for antigen-specific T-cell cytokine expression. The impact on muscle was measured by grip strength assessment, in vivo torque, optical imaging for inflammation and H&E staining of sections to assess muscle damage.

Results:

We found that AAV-9-microdystrophin gene therapy induced expression of microdystrophin, anti-dystrophin antibodies, and T-cell cytokine responses. Immunomodulatory treatments, rituximab and VBP6 completely abrogated the anti-dystrophin antibody response. Prednisolone, CTLA4-Ig, and eplerenone showed variable efficacy in blocking the anti-dystrophin immune response. In contrast, none of the drugs completely abrogated the antigen specific IFN-γ response. AAV-microdystrophin treatment significantly reduced inflammation in both forelimbs and hindlimbs, and the addition of prednisolone and VBP6 further reduced muscle inflammation. Treatment with immunomodulatory drugs, except eplerenone, enhanced the beneficial effects of AAV-microdystrophin therapy in terms of force generation.

Conclusions:

Our data suggest that AAV-microdystrophin treatment results in anti-dystrophin antibody and T-cell responses, and immunomodulatory treatments have variable efficacy on these responses.

INTRODUCTION

Dystrophin correction and restoration strategies such as exon skipping and gene therapy have shown significant potential for treating patients with Duchenne muscular dystrophy (DMD). There is evidence in both DMD patients and animal models that dystrophin replacement by gene therapy leads to an anti-dystrophin immune response [1, 2] that is likely to limit the long-term use of these therapeutic strategies as has been seen in many other disorders [3]. AAV vectors are known for their low immunogenicity, which leads to vector persistence and long-term transgene expression. It is proposed that the reduced ability of AAV vectors to activate antigen-presenting cells may account for their decreased immunogenicity [4]. It is also well known that AAV vectors elicit cellular and humoral immune responses directed against the viral capsid and the transgene product. These immune responses affect clinical outcomes; therefore it is essential to study the mechanisms that lead to the induction of immune responses, particularly against transgenes [5]. Yuasa et al. have shown that AAV vector administration using the muscle-specific MCK promoter elicits an immune response to the transgene in dystrophin-deficient mice, suggesting that neo-antigens introduced by AAV vectors can evoke immune reactions in mdx muscle [6]. It has also been shown that rAAV-expressing alpha-sarcoglycan can cause a dramatic loss of positive fibers less than 1.5 months after rAAV injection due to cytotoxic effects mediated against the alpha-sarcoglycan transgene [7]. Finally, it is also well documented that gene transfer of human full-length or mini-dystrophin provokes both humoral and cytotoxic responses, leading to the destruction of the transfected fibers [8]. Here, we tested the hypothesis that the pro-inflammatory milieu of dystrophic muscle augments immune responses against de novo dystrophin. This response can be reduced using immunomodulatory treatments that target T cells, B cells, and pro-inflammatory processes. We have selected CTLA4-Ig, a soluble fusion protein consisting of the extracellular domain of CTLA-4 linked to the Fc portion of mouse IgG2a that interferes with T-cell co-stimulation by inhibiting the CD28-B7 interaction. The targeting of co-stimulatory signals prevents autoimmune responses because it only affects T cells undergoing activation [9, 10]. Similarly, we targeted B cells with rituximab, a chimeric murine/ human monoclonal antibody targeted against the B-cell marker, CD20. Rituximab binding initiates a cascade of intracellular signals that result in rituximab-mediated B-cell killing [11]. Rituximab is currently FDA approved for use in rheumatoid arthritis and non-Hodgkin’s lymphoma and is therefore readily available for DMD patients. We targeted the inflammatory process using three drugs (prednisolone, VBP6, and eplerenone) because of their known anti-inflammatory properties [12–14]. Using this approach we were able to systematically evaluate the effects of the above immunomodulatory and anti-inflammatory drugs not only on dystrophin expression and anti-dystrophin immune response, but also on muscle inflammation, muscle damage, and muscle function.

MATERIALS AND METHODS

Animal preclinical studies

All mouse experiments were performed in accordance with the Institutional Animal Care and Use Committee (IACUC) guidelines at Binghamton University. Five-week old male wild type (C57BL/10ScSn/J) and mdx (C57BL/10ScSn-Dmd<mdx> /J) mice were obtained from The Jackson Laboratory (Bar Harbor, ME, USA) and housed at the Binghamton University animal facility under 12 h light/dark cycle. Unless otherwise noted, standard mouse chow and water were provided ad libitum. Mice were permitted a one-week acclimation period prior to the start of experiments.

Administration of the AAV9 expressing microdystrophin and immune-modulators

rAAV was manufactured by the Viral Vector Core at University of Massachusetts Medical School using triple transfection of adherent HEK293 cells followed by ultracentrifugation purification. AAV titer was determined by qPCR. mdx mice were randomized into 7 groups (15/group): group-1, no treatment control; group-2, AAV-microdystrophin alone; group-3, AAV-microdystrophin + CTLA4-Ig; group-4, AAV-microdystrophin + rituximab; group-5, AAV-microdystrophin + prednisolone; group-6, AAV-microdystrophin + VBP6; group-7, AAV-microdystrophin + eplerenone. C57BL/10 mice served as wild type control (group-8). rAAV9-microdystrophin (μDysH2) was obtained from Solid Biosciences and administered to six week old mice via tail vein injection as a single dose at 1×1014 vg/kg. CTLA4-Ig (AdipoGen® Life Sciences, San Diego, CA, USA) was administered intraperitoneally (I.P.) every two weeks at 0.5 mg/kg body weight. Rituximab (Genentech,USA, a kind gift from Children’s National Medical Center, Washington DC) was administered I.P. every four weeks at 1.0 mg/kg body weight. VBP6 (Reveragen Biopharma, Rockville, MD, USA) and prednisolone (Sigma-Aldrich, St. Louis, MO, USA) were administered daily as an oral suspension in cherry syrup (HUMCO ™, Austin, TX, USA) at 15 mg/kg and 5 mg/kg body weight respectively. Eplerenone (Beijing Cooperate Pharmaceutical Co. Ltd., Beijing, China) containing chow (Envigo Teklad Diets, Madison, WI, USA) was formulated at 2000 mg/kg to deliver a daily dose of 200 mg/kg body weight. Grip strength, optical imaging and isometric torque measurements began after 12 weeks of treatment and administration of immune-modulators continued until the completion of these assessments (Fig. 1). Weekly body weights were recorded for all mice and expressed in grams for absolute values and as percentage weight gained after 12 weeks of treatment.

Fig. 1

Experimental Design. Six-week old mice treated with AAV-microdystrophin (AAV-μDys) at week zero.

Experimental Design. Six-week old mice treated with AAV-microdystrophin (AAV-μDys) at week zero.

Grip strength test

Grip strength was assessed using a grip strength meter (GSM) consisting of horizontal forelimb mesh and force gauge (Columbus Instruments, Columbus, OH, USA). All mice were acclimatized on forelimb meshes for 5 consecutive days one week prior to actual data collection. For forelimb strength assessment, the animal was allowed to hold the horizontal mesh with the forelimb paws and then was gently pulled back until its grip was broken. The force transducer retained the peak force reached when the animal’s grip was broken and is shown on a digital display. This was repeated five times during each session within a 2-minute time-frame. The maximum values for each day over a 5-day period were used for subsequent analysis. For both acclimatization and data collection the test was performed in a blinded fashion by the same person and at the same time of day. This data is expressed as normalized forelimb strength kgF/kg.

Isometric Torque

An in vivo procedure was used that allows for non-invasive assessment of dorsiflexor muscle function by fibular (also known as peroneal) nerve stimulation in mice. All force measurements were performed with 1300A: 3-in-1 Whole Animal System (Aurora Scientific Inc. Aurora, ON, Canada).

First, the mouse was placed into the anesthesia chamber with an oxygen flow rate of 1 L/min with 5%isoflurane (via nosecone inhalation) until the mouse lost consciousness. Once adequate anesthesia was confirmed via loss of the foot reflex, all hair on the left leg of the mouse was removed by shaving with electric hair clippers. The mouse was then placed in a supine position on the heated platform and the foot was placed onto the footplate and attached using medical tape. The knee was clamped to stabilize and immobilize the leg during the procedure and the platform adjusted so that there was a 90° angle at the ankle. Electrodes were placed on the lateral side of the right leg; one near the head of the fibula and the other electrode more distally on the lateral side of the leg (subcutaneously).

The high-power bi-phase stimulator was adjusted as needed to obtain a stimulation of the peroneal nerve that results in maximum dorsiflexion torque. During stimulation, the transducer was turned clockwise to yield negative values, which are important to ensure that the electrodes are stimulating only the dorsiflexor muscles by peroneal nerve. Once this step was achieved, electrodes were stabilized using a clamp, to prevent any movement during the procedure. The stimulation was performed at various incremental frequencies (20, 40, 60, 80, 100, 120, 140, 160, 180 and 200 Hz) (Aurora Scientific, Aurora, ON, Canada). All tests were performed in a blinded fashion and the data is expressed as normalized isometric torque (mN-m/g).

Optical imaging of muscle using Cathepsin-B

Quantification of macrophage activity in forelimb and hindlimb muscles of mice was achieved using live-animal optical imaging of Cathepsin-B (CTSB) enzyme activity using a caged near-infrared substrate (ProSense 680) as previously described [15]. Briefly, mice underwent retro-orbital injections with 1.5 nmol ProSense 680 (Perkin–Elmer, Waltham, MA, USA) in PBS 24 h prior to imaging. Mice were anaesthetized with isoflurane, and hair from areas to be scanned was removed with Nair. Imaging was performed in an IVIS® Spectrum in vivo imaging system (Perkin–Elmer), with the floor of the imager heated to maintain physiological body temperature and mice kept anaesthetized by administration of 2%isoflurane via a nose cone. Cathepsin activity was measured from forelimb and hindlimb muscles and defined as intensity in terms of photon counts/cm2. Intensity was calculated using Living Image software for a defined area of interest. All tests were performed in a blinded fashion and data expressed as average radiant efficiency ([p/s/cm2/sr]/[μW/cm2]).

Tissue and serum collection

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. Blood for serum was also collected from the submandibular vein prior to and approximately 6 weeks after the first treatment. Tissues (muscles and organs) were surgically removed at the time of necropsy and weighed. Halved organs (heart, diaphragm) and paired skeletal muscles were either stored in formalin or flash-frozen in liquid nitrogen-chilled isopentane. All frozen serum and tissue samples were stored at –80°C for analysis.

Hematoxylin and Eosin (H&E) staining and dystrophic severity scoring

Isopentane-frozen quadriceps muscle specimens were sectioned at 8-micron thickness and stained with H&E using a Sakura Tissue-Tek Prisma Automated Slide Stainer available through the Children’s Hospital of Wisconsin Research Institute’s Histology Core Facility. These slides were assessed in a blinded fashion by a board-certified neuropathologist with respect to findings associated with active dystrophic disease, including myofiber degeneration, active myofiber regeneration (basophilic fibers), and inflammation. The area of each sample displaying these findings was visually estimated and the following grading scheme was applied: Grade 0 = normal, grade 1 = chronic regenerative changes only, grade 1.5 = very mild (<5%of muscle area with active dystrophic pathology), grade 2 = mild (6–20%of muscle area with active dystrophic pathology), grade 2.5 = mild to moderate (21–30%of muscle area with active dystrophic pathology), grade 3 = moderate (30–50%of muscle area with active dystrophic pathology), grade 3.5 = moderate to severe (51–60%of muscle area with active dystrophic pathology) and grade 4 = severe (>60%of muscle area with active dystrophic pathology). Areas indicative of chronic regeneration (internally nucleated fibers with appropriate eosinophilia, endomysial fibrosis, or fatty infiltration) were not integrated into the grading of dystrophic severity, as it is possible that the damage associated with these changes occurred before treatment.

Flow cytometry for immunophenotyping

Terminal peripheral blood was obtained via cardiac puncture and red blood cells lysed by incubation in RBC lysis buffer (BioLegend, San Diego, CA, USA). Lymphocyte staining was performed with monoclonal antibodies PE anti-CD45 (Cat. No. 103106), APC anti-CD4 (Cat. No.100411), APC anti-CD8 (Cat. No. 100711), APC anti-CD11b (Cat. No.101212) or APC anti-CD20 (Cat. No. 150411) purchased from Biolegend. Flow cytometry was performed on a BD Accuri™ C6 Plus (BD Biosciences, San Jose, CA). The data is expressed as percentage of CD45+ cell population.

Western blot to assess antibody response to dystrophin

To identify antibodies generated by the mdx mice against the human microdystrophin treatment, total protein was extracted from dystrophin sufficient human skeletal muscle (kindly provided by Eric Hoffman) using lysis buffer (100 mM Tris-HCl pH 8.0, 5%SDS) containing protease inhibitors (HALT™ Protease Inhibitor Cocktail 100x, Thermo Fisher Scientific, USA). Proteins (50 ug/lane) were resolved through NuPage™ Tris-Acetate 3–8%gels (Thermo Fisher Scientific) and electro-blotted overnight at 4°C onto nitrocellulose membranes. To assess the equality of transfer, membranes were stained with Ponceau S (Thermo Fisher Scientific, USA) and subsequently cut into individual strips. After blocking for 1 hour in 5%milk/PBST (0.1%), each strip was incubated overnight at 4°C with a 1:150 dilution of the mouse serum of interest. As a positive control the presence of dystrophin protein was confirmed by incubating one strip per blot with GTX15277 anti-dystrophin antibody (GeneTex Inc., Irvine, CA, USA). Blots were washed and incubated with anti-mouse horseradish peroxidase (HRP) conjugated secondary antibody (1:5000) for mouse autoantibodies and anti-rabbit HRP conjugated secondary antibody (1:5000) for GTX15277 anti-dystrophin. The chemiluminescent signal was detected with ECL reagent (Amersham Biosciences). The data is expressed as positive or negative for detection of anti-dystrophin antibodies.

Mass spectrometry assay for microdystrophin quantification

Muscle tissues were cryostat sliced, extracted and total protein determined by BCA (ThermoFisher Scientific). Equal protein aliquots were spiked with an absolute quantity of an isotopically labeled dystrophin peptide and samples digested with trypsin. The labeled and unlabeled dystrophin peptide pairs were immune-enriched using a peptide-specific antibody and then analyzed by LC-MS/MS. The intensity of the heavy to the light peptide signal were used to calculate the fmol of dystrophin in each sample. The data is expressed as average fmol and percentage of average fmol of dystrophin detected in wild type mice.

Immunofluorescence for microdystrophin quantification

Isopentane-frozen quadriceps muscle specimens were sectioned at 8-micron thickness and immunostained per standard techniques for dystrophin/microdystrophin (Developmental Studies Hybridoma Bank (DSHB), MANEX44A). Evaluation of percent dystrophin or microdystrophin positive fibers was performed in a blinded fashion by a neuropathologist (M.W.L.) using a Zeiss AxioImager Z1 fluorescent microscope and estimated to the nearest 5%.

Antigen specific T-cell cytokine responses to dystrophin in spleen cells

Spleens were surgically removed, transferred to a culture dish and injected with PBS to remove red blood cells and lymphocytes. To disturb the compact germinal center, spleens were rubbed between the rough surface of glass slides until they dissipated The tissue was then passed through a cell strainer and cells collected by centrifugation. Red blood cells were removed using RBC lysis buffer and cells re-suspended in RPMI 1640 before counting and storage in liquid nitrogen.

For T cell stimulation, splenocytes were thawed and plated in 96-well plates at 5.0×106 cells/well. Splenocytes were either unstimulated, stimulated with a cocktail of three dystrophin-derived peptides ((Peptide 1: TSWSDGLALNALIHSHRPDL; Peptide2: TYPDKKSILMYITSLFQVLP; Peptide 3: ELDKLRQAEVIKGSWQPVG) at a final concentration of 10μg/ml for each, treated with scrambled control peptide (Peptide 4: TSWRDGLAFNALIHSHRPDL) or stimulated with Dynabeads Mouse T-Activator CD3/CD28 (BD Cat. No.11456D) as a positive control for 48 hours.

After stimulation, culture supernatants were collected, and assays for the cytokines IFN-γ and IL-2 were performed using the BD™ CBA Mouse Enhanced Sensitivity Flex Set System according to the manufacturer’s instructions (BD™ Cytometric Bead Array (CBA) Mouse Enhanced Sensitivity Master Buffer Kit BD Cat. No.562262, Mouse IL-2 Enhanced Sensitivity Flex Set Cat. No.562262, Mouse IFN-γ Enhanced Sensitivity Flex Set Cat. No.562233). These assays employ particles with discrete fluorescence intensities to detect soluble analytes at very low concentrations. BD Accuri™ C6 Plus Flow Cytometer and the software that comes with the instrument were used to collect and analyze data.

For quantification of T cell response from each mouse, we calculated the ratio of cytokine concentration of the cell supernatant treated with the peptide cocktail to the untreated control group. The cutoff ratio was calculated by the mean + 2 standard deviations of the ratio of cytokine concentration of peptide treated/untreated control from each mouse of the mdx only group. The positive T-cell response cutoff for IFN-gamma and IL-2 was 1.44 and 1.43 respectively. Fisher exact test was used to calculate the difference between the mdx only group with the AAV and immunomodulatory drug-treated groups.

Statistical analysis

A two-tailed Student’s t test was used for experiments involving only two groups. For experiments involving more than two groups, one-way ANOVA with Dunnett’s post-test comparison against a control was used. For binary data (positive/negative) Fischer’s exact test was used. Significance was defined as a p-value less than 0.05. All data is expressed as mean±standard error of the mean (S.E.M.). Prism statistical software (GraphPad, La Jolla, CA) was used for statistical analyses.

RESULTS

Effects on body weights

mdx mice weighed significantly more than the wild type mice at 15 weeks. Weight gain was significantly reduced in AAV-microdystrophin treated mdx mice in comparison to untreated mdx mice (Table 1, Fig. 2). Addition of immunomodulatory drugs further reduced body weight gain but this was only statistically significant for prednisolone and VBP6. This reduction was highly significant for prednisolone (p < 0.0001), consistent with the known catabolic activity of this drug.

Table 1

Comparison of body weight after 15 weeks of treatment

WTmdxmdx + AAV-μDysmdx + AAV-μDys CT-Igmdx + AAV-μDys RTXmdx + AAV-μDys PRDmdx + AAV-μDys VBP6mdx + AAV-μDys EPL
Body weight(g)28.0529±0.381333.9286±0.4422§§§§34.4025±0.540234.2528±0.542333.8394±0.424629.0214±0.5358****31.9447±0.7383**33.6517±0.5610
Body weight gain (%) after 15wks35.6331±2.404250.5470±3.7403§§37.4684±2.4729#36.1823±1.494336.3494±1.813816.5701±1.4513****29.9543±1.9963*32.5642±2.2067

Symbols for significant differences are: §=between mdx and WT, #=between mdx treated with AAV-microdystrophin (AAV-μDys) and mdx without treatment and * = between immunosuppressive drug treated mdx and mdx AAV-μDys. Data presented as mean±standard error of measurement (SEM). *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001. AAV- μDys AAV-microdystrophin, CT-Ig CTLA4-Ig, RTX rituximab, PRD prednisolone, EPL eplerenone.

Fig. 2

Body weight measurements during treatment. Body weight was recorded each week over the entire duration of treatment. Data presented as mean±SEM. AAV- μDys AAV-microdystrophin, CT-Ig CTLA4-Ig, RTX rituximab, PRD prednisolone, EPL eplerenone.

Body weight measurements during treatment. Body weight was recorded each week over the entire duration of treatment. Data presented as mean±SEM. AAV-
μDys AAV-microdystrophin, CT-Ig CTLA4-Ig, RTX rituximab, PRD prednisolone, EPL eplerenone.

Effects on tissue weights

Tissue weights were normalized to body weight at sacrifice. TA, EDL, quadriceps and triceps normalized weights were significantly higher in mdx than in wild type mice. TA, quadriceps and triceps normalized weights were significantly reduced in the AAV-microdystrophin treated mdx mice in comparison to untreated mdx mice. Addition of immunomodulatory drugs did not alter muscle weights in comparison to AAV-microdystrophin treated mdx mice with the exception of significant reduction in TA weight following prednisolone treatment (Table 1).

Spleen weight was significantly higher in AAV-microdystrophin treated mice in comparison to untreated mice. Treatment with rituximab, prednisolone, VBP6 or eplerenone significantly reduced spleen weight (Table 1). This decrease was highly significant for prednisolone treated mice (p < 0.0001), consistent with known immunosuppressive properties of this drug.

Normalized heart weight was reduced in mdx compared to wild type mice. We did not see significant differences in heart weights between mdx and AAV-microdystrophin treated mdx mice and this was unaffected by immunomodulatory drugs (Table 1).

Effects on grip strength

Since there are differences in body weights between normal and mdx mice, we have normalized forelimb strength to weight. Normalized forelimb grip strength in mdx mice is significantly lower than in wild type controls. Treatment with AAV-microdystrophin significantly increased the forelimb grip strength force (Fig. 3).

Fig. 3

Grip strength measurement results of wild type and mdx mice with different treatments. Grip strength measurements were performed after 12 weeks of treatment and normalized to body weight. Data presented as mean±SEM. *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001. AAV- μDys AAV-microdystrophin, CT-Ig CTLA4-Ig, RTX rituximab, PRD prednisolone, EPL eplerenone.

Grip strength measurement results of wild type and mdx mice with different treatments. Grip strength measurements were performed after 12 weeks of treatment and normalized to body weight. Data presented as mean±SEM. *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001. AAV-
μDys AAV-microdystrophin, CT-Ig CTLA4-Ig, RTX rituximab, PRD prednisolone, EPL eplerenone.

Addition of immunomodulatory treatments significantly increased forelimb grip strength force compared to treatment with AAV-microdystrophin alone, with the exception of eplerenone which did not show any difference. Increases in grip strength were highly statistically significant for prednisolone and VBP6. This data suggests that, with the exception of eplerenone, addition of immunomodulatory drugs enhances the beneficial effects of AAV-microdystrophin treatment in terms of force generation.

Effects on peripheral blood mononuclear cell phenotype

CD4 T-cells were significantly increased in the peripheral blood of mdx mice in comparison to wild type controls (Fig. 4A). Treatment with AAV-microdystrophin did not alter the percentage of CD4 T-cells in comparison to untreated mice. However, addition of rituximab or prednisolone significantly decreased CD4 T-cell counts compared to AAV-microdystrophin treated mice. Treatment with other immunomodulatory drugs did not significantly alter CD4 T-cell levels.

Fig. 4

Representative flow cytometry analysis of peripheral blood leukocytes from mice treated with combination of AAV-microdystrophin with immunosuppressive drug. A,percentage of CD4 + cells in CD45 + cells; B, percentage of CD4 + cells in CD45 + cells;C, percentage of CD4 + cells in CD45 + cells D, percentage of CD4 + cells in CD45 + cells. Data presented as mean±SEM. *P≤0.05, **P≤0.01. AAV- μDys AAV-microdystrophin, CT-Ig CTLA4-Ig, RTX rituximab, PRD prednisolone, EPL eplerenone.

Representative flow cytometry analysis of peripheral blood leukocytes from mice treated with combination of AAV-microdystrophin with immunosuppressive drug. A,percentage of CD4 + cells in CD45 + cells; B, percentage of CD4 + cells in CD45 + cells;C, percentage of CD4 + cells in CD45 + cells D, percentage of CD4 + cells in CD45 + cells. Data presented as mean±SEM. *P≤0.05, **P≤0.01. AAV-
μDys AAV-microdystrophin, CT-Ig CTLA4-Ig, RTX rituximab, PRD prednisolone, EPL eplerenone.

CD8 T-cells were significantly increased in mdx mice in comparison to wild type controls (Fig. 4B). However, neither AAV-microdystrophin or immunomodulatory treatment significantly altered the CD8 T-cell population.

We did not detect significant differences in CD11b positive leukocytes between wild type controls, mdx or mdx AAV-microdystrophin treated groups. However, we found a significant increase in CD11b positive cells in peripheral blood after treatment with prednisolone (Fig. 4C).

We did not detect significant differences in CD20 positive B-cells between wild type controls, mdx or mdx AAV-microdystrophin treated groups. However, addition of CTLA4-Ig significantly increased and addition of prednisolone significantly reduced the number of CD20 positive B-cells. Addition of rituximab, VBP6 or eplerenone trended towards reduction, although they did not reach statistical significance, consistent with the immunosuppressive properties of these treatments [16, 17].

Effects on Cathepsin-B activity in muscle

Cathepsin-B imaging probes sensitive in the near infrared fluorescence spectrum were used. Cathepsin-B in macrophages cleaves the probes to emit fluorescence where macrophage activity is heightened. We have measured activity in terms of average radiant efficiency ([p/s/cm2/sr]/[μW/cm2]).

Cathepsin-B activity was significantly increased in mdx mice in comparison to wild type controls. AAV-microdystrophin treatment significantly reduced cathepsin-B activity in both forelimbs and hindlimbs (Fig. 5A, B) and addition of prednisolone and VBP6 further reduced cathepsin-B activity in muscle. Addition of CTLA-Ig, rituximab or eplerenone did not significantly alter cathepsin-B activity in AAV-microdystrophin treated mdx mice.

Fig. 5

Analysis of muscle inflammation using optical imaging of cathepsin. Cathepsin activity was measured from A) forelimb and B) hindlimb muscles as a quantification of inflammation. (C) Whole body IVIS images of treated mice. Activity is defined in terms of average radiant efficiency [p/s/cm2/sr]/[μW/cm2]. Data presented as mean±SEM. *P≤0.05, **P≤0.01. AAV- μDys AAV-microdystrophin, CT-Ig CTLA4-Ig, RTX rituximab, PRD prednisolone, EPL eplerenone.

Analysis of muscle inflammation using optical imaging of cathepsin. Cathepsin activity was measured from A) forelimb and B) hindlimb muscles as a quantification of inflammation. (C) Whole body IVIS images of treated mice. Activity is defined in terms of average radiant efficiency [p/s/cm2/sr]/[μW/cm2]. Data presented as mean±SEM. *P≤0.05, **P≤0.01. AAV-
μDys AAV-microdystrophin, CT-Ig CTLA4-Ig, RTX rituximab, PRD prednisolone, EPL eplerenone.

Effects on isometric torque

AAV-microdystrophin treatment significantly increased normalized isometric torque in comparison to untreated mdx mice. Immunomodulatory drugs, with the exception of eplerenone, significantly improved normalized torque compared to AAV-microdystrophin alone, this improvement was highly significant for addition of prednisolone and VBP6 (p < 0.001) (Fig. 6).

Fig. 6

Isometric torque of dorsiflexor muscles of mice treated with combination of AAV-microdystrophin with immunosuppressive drug. Isometric force produced at frequencies 200 Hz was plotted. Data presented as mean±SEM. *P≤0.05, **P≤0.01, ***P≤0.001. AAV- μDys AAV-microdystrophin, CT-Ig CTLA4-Ig, RTX rituximab, PRD prednisolone, EPL eplerenone.

Isometric torque of dorsiflexor muscles of mice treated with combination of AAV-microdystrophin with immunosuppressive drug. Isometric force produced at frequencies 200 Hz was plotted. Data presented as mean±SEM. *P≤0.05, **P≤0.01, ***P≤0.001. AAV-
μDys AAV-microdystrophin, CT-Ig CTLA4-Ig, RTX rituximab, PRD prednisolone, EPL eplerenone.
Table 2

Comparison of normalized tissue weights after treatment

WTmdxmdx + AAV-μDysmdx + AAV-μDys CT-Igmdx + AAV-μDys RTXmdx + AAV-μDys PRDmdx + AAV-μDys VBP6mdx + AAV-μDys EPL
TA0.1608±0.00600.2088±0.0061§§§§0.1832±0.0028# # #0.1889±0.00330.1806±0.00250.1940±0.0045*0.1947±0.00520.1866±0.0057
EDL0.0336±0.00220.0416±0.0019§0.0389±0.00070.0401±0.00100.0391±0.00120.0419±0.00150.0390±0.00080.0415±0.0012
Quad0.6235±0.02550.8463±0.0308§§§§0.7695±0.0118# #0.7729±0.01170.7524±0.01130.7428±0.02360.7411±0.01120.7617±0.0176
Gastroc0.5479±0.00890.5479±0.00650.5624±0.00700.5733±0.00690.5616±0.00470.5655±0.00590.5731±0.00720.5706±0.0196
Soleus0.0341±0.00140.0356±0.00130.0329±0.00110.0334±0.00100.0338±0.00080.0329±0.00120.0350±0.00070.0345±0.0010
Tricep0.3803±0.01620.6002±0.0109§§§§0.4850±0.0122# # # #0.5102±0.00940.5057±0.00820.5037±0.01010.4703±0.02970.5074±0.0096
Spleen0.2953±0.00780.2705±0.01290.2991±0.0061#0.2859±0.00840.2770±0.0082*0.2143±0.0056****0.2727±0.0080*0.2714±0.0072**
Heart0.5605±0.01700.4841±0.0188§§0.5037±0.01460.5254±0.02190.5162±0.01600.4961±0.01680.5128±0.02230.5079±0.0154

Tissue weights as percentage of body weight at sacrifice (grams). Symbols for significant differences are: §= between mdx and WT, #= between mdx treated with AAV-microdystrophin (AAV-μDys) and mdx without treatment, and * = between immunosuppressive drug treated mdx and mdx AAV-μDys. Data presented as mean±standard error of measurement (SEM). *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001. AAV- μDys AAV-microdystrophin, CT-Ig CTLA4-Ig, RTX rituximab, PRD prednisolone, EPL eplerenone.

Effects on anti-dystrophin antibodies

We have evaluated the anti-dystrophin antibody response using western blotting. We found two out of 10 (20%) untreated animals showed antibodies to dystrophin, whereas 11 out of 19 (58%) of AAV-microdystrophin treated animals showed anti-dystrophin antibodies (Table 3). A representative western blot image as well as densitogram peaks showing the antibody positive sera is shown in Fig. 7A and B.

Table 3

Dystrophin reactive antibody positivity per group

Treatment GroupTestedPositive%P-value (Fisher’s exact test)
mdx only10220
mdx + AAV-μDys191157.80.1142
mdx + AAV-μDys + CT-Ig164250.0866
mdx + AAV-μDys + RTX16000.0002
mdx + AAV-μDys + PRD1417.10.0036
mdx + AAV-μDys + VBP616000.0002
mdx + AAV-μDys + EPL18422.20.0448

Western blot analysis for the presence of dystrophin-reactive antibodies in mdx mice treated with AAV-microdystrophin (AAV-μDys). Protein extract from dystrophin sufficient human skeletal muscle was incubated with terminal serum (1:150) collected from mdx mice injected with a single dose of AAV-microdystrophin or vehicle. Commercial antibody is used as positive control antibody to detect dystrophin. AAV- μDys AAV-microdystrophin, CT-Ig CTLA4-Ig, RTX rituximab, PRD prednisolone, EPL eplerenone.

Fig. 7

Western blot and densitometry analysis demonstrating the presence of dystrophin-reactive antibodies in mdx mice treated with AAV-microdystrophin alone. Protein extract from dystrophin sufficient human skeletal muscle was incubated with terminal serum (1 : 150) collected from mdx mice injected with a single dose of AAV-microdystrophin (AAV1-AAV15) or vehicle (C1, C2). Commercial antibody is used as positive control for dystrophin detection. AAV- μDys AAV-microdystrophin, CT-Ig CTLA4-Ig, RTX rituximab, PRD prednisolone, EPL eplerenone.

Western blot and densitometry analysis demonstrating the presence of dystrophin-reactive antibodies in mdx mice treated with AAV-microdystrophin alone. Protein extract from dystrophin sufficient human skeletal muscle was incubated with terminal serum (1 : 150) collected from mdx mice injected with a single dose of AAV-microdystrophin (AAV1-AAV15) or vehicle (C1, C2). Commercial antibody is used as positive control for dystrophin detection. AAV-
μDys AAV-microdystrophin, CT-Ig CTLA4-Ig, RTX rituximab, PRD prednisolone, EPL eplerenone.

Treating animals with immunomodulatory drugs significantly reduced the frequency of antibody positive animals with the exception of CTLA4-Ig treatment. However, the magnitude of reduction significantly varied between immunomodulatory agents. For example, rituximab and VBP6 showed 0%positivity (p < 0.001) for anti-dystrophin antibodies, whereas prednisolone and eplerenone showed 7%(p < 0.01) and 22%(p < 0.05) positivity respectively (Table 3).

Effects on anti-dystrophin T-cell response

We synthesized three T cell peptides that span the N-terminal portion of microdystrophin (P17, P19, P23) and one C-terminal peptide in Hinge 4 (P74) based on a paper published by Mendell et al. [1]. It is pertinent to note that we have used a human dystrophin gene therapy construct in our mouse experiments. Locations of the T cell epitopes on full length dystrophin and micro DysH2 are illustrated in Fig. 8 [18]. Our data indicated that AAV-microdystrophin treated mice show a dystrophin specific T cell response (IFN-γ production) (Fig. 8, Table 4). We observed a significant difference in IFN-γ production stimulated by CD3/CD28 and dystrophin peptides. The frequency of antigen specific T cells in the spleen is very low [19], whereas 100%of T cells express CD3/CD28 resulting in a response several orders of magnitude higher for CD3/CD28 stimulation. Not all mice showed T cell responses, which is consistent with our previous observation of anti-dystrophin antibody and T cell responses after PMO mediated exon skipping treatment [20] in dystrophin deficient mdx mice.

Fig. 8

Dystrophin specific T-cell responses in mdx mice treated with AAV-microdystrophin. A. Full length human dystrophin showing various domains. B. Micro dystrophin H2 showing N terminal actin binding domain, H1, rod domains 1, 2, 3, H2, rod domain 24, H4 and cystine rich domains. T cell epitopes are marked with purple rectangles with full length amino acid numbers on either side of the rectangle. C. Representative FACS image of antigen specific IFN-γ production in spleen cells of AAV treated mdx mice. Splenocytes (2×106 cells/well) isolated from AAV treated mdx mice were incubated for 24hrs with No peptides (electric blue), mixture of dystrophin peptides (50ug/ml) (P19: FDWNSVVCQQSATQRLEHAF; P17: TSWSDGLALNALIHSHRPDL; p23: TYPDKKSILMYITSLFQVLP p74: ELDLKLRQAEVIKGSWQPVG (royal blue), scrambled control peptide,50 ug/ml (TSWRDGLAFNALIHSHRPDL) and CD3/CD28 beads (pumpkin). Supernatants were collected and assayed using BD™ Cytometric Bead Array.

Dystrophin specific T-cell responses in mdx mice treated with AAV-microdystrophin. A. Full length human dystrophin showing various domains. B. Micro dystrophin H2 showing N terminal actin binding domain, H1, rod domains 1, 2, 3, H2, rod domain 24, H4 and cystine rich domains. T cell epitopes are marked with purple rectangles with full length amino acid numbers on either side of the rectangle. C. Representative FACS image of antigen specific IFN-γ production in spleen cells of AAV treated mdx mice. Splenocytes (2×106 cells/well) isolated from AAV treated mdx mice were incubated for 24hrs with No peptides (electric blue), mixture of dystrophin peptides (50ug/ml) (P19: FDWNSVVCQQSATQRLEHAF; P17: TSWSDGLALNALIHSHRPDL; p23: TYPDKKSILMYITSLFQVLP p74: ELDLKLRQAEVIKGSWQPVG (royal blue), scrambled control peptide,50 ug/ml (TSWRDGLAFNALIHSHRPDL) and CD3/CD28 beads (pumpkin). Supernatants were collected and assayed using BD™ Cytometric Bead Array.
Table 4

FN-γ and IL-2 secretion after treatment of splenocytes with dystrophin peptides

IFN-γIL-2
Treatment GroupTestedPositive%Positive%
mdx only1000.0%00.0%
mdx + AAV-μDys18633.3%527.8%
mdx + AAV-μDys + CT-Ig18633.3%422.2%
mdx + AAV-μDys + RTX16318.8%16.3%
mdx + AAV-μDys + PRD1317.7%17.7%
mdx + AAV-μDys + VBP617317.6%423.5%
mdx + AAV-μDys + EPL17847.1%00.0%

Positive if ratio of peptide treated to untreated cells cytokine response was greater than the mean+2 standard deviations of the mdx only group. AAV- μDys AAV-microdystrophin, CT-Ig CTLA4-Ig, RTX rituximab, PRD prednisolone, EPL eplerenone.

Table 4 shows that the percentage of mice with dystrophin specific T cell responses was reduced after rituximab, prednisolone and VBP6 in comparison to eplerenone and CTLA4-Ig treated mice, suggesting that suppressing T and B cell responses is unlikely to completely abolish cell mediated immunity after rAAV treatment. However, none of these changes were statistically significant by Fishers exact test. No T cell responses were detected in BL10 or in untreated mdx mice.

Effects on dystrophin expression

Mass spectrometry analysis showed that treatment with AAV-microdystrophin increased dystrophin levels to nearly 80%of normal. With the exception of CTLA4-Ig, all immunomodulatory treatments showed percentages above the AAV-microdystrophin only group but none of them reached statistical significance (Table 5).

Table 5

Dystrophin expression detected by mass spectrometry

Treatment GroupTestedAverage fmolSDCV%of Normalp-value (t-test)
mdx only3000
mdx + AAV-μDys887.024.728.479.80.0002
mdx + AAV-μDys + CT-Ig882.827.333.075.90.7505
mdx + AAV-μDys + RTX898.341.342.090.10.5191
mdx + AAV-μDys + PRD997.323.824.489.20.3946
mdx + AAV-μDys + VBP69104.324.423.495.60.1672
mdx + AAV-μDys + EPL8103.021.120.594.40.2057

Average fmol of dystrophin, standard deviation (SD), coefficient of variation (CV) and %of dystrophin compared to wild type mice measured by mass spectrometry. P-values calculated by unpaired T-test for mdx only compared to mdx treated with AAV-microdystrophin (mdx + AAV-μDys) group and then mdx + AAV-μDys group with each immunomodulatory drug group. AAV- μDys AAV-microdystrophin, CT-Ig CTLA4-Ig, RTX rituximab, PRD prednisolone, EPL eplerenone.

Treatment with AAV-microdystrophin increased dystrophin detected by immunofluorescence to over 75%of wild type levels (Fig. 9). Addition of immunomodulatory drugs made no significant change to the level of immunofluorescence detected.

Fig. 9

Dystrophin detected by immunofluorescence. Percentage of dystrophin or microdystrophin positive fibers in quadriceps sections detected by immunostaining (Developmental Studies Hybridoma Bank (DSHB), MANEX44A) using a Zeiss AxioImager Z1 fluorescent microscope and estimated to the nearest 5%. Data presented as mean±SEM. AAV- μDys AAV-microdystrophin, CT-Ig CTLA4-Ig, RTX rituximab, PRD prednisolone, EPL eplerenone.

Dystrophin detected by immunofluorescence. Percentage of dystrophin or microdystrophin positive fibers in quadriceps sections detected by immunostaining (Developmental Studies Hybridoma Bank (DSHB), MANEX44A) using a Zeiss AxioImager Z1 fluorescent microscope and estimated to the nearest 5%. Data presented as mean±SEM. AAV-
μDys AAV-microdystrophin, CT-Ig CTLA4-Ig, RTX rituximab, PRD prednisolone, EPL eplerenone.

Dystrophic pathology on H&E stained sections

In comparison to untreated mdx mice, AAV-microdystrophin treated mice showed a lower dystrophic severity score, which corresponds to a lower proportion of the quadriceps muscle in which active myofiber degeneration, regeneration, or inflammation could be observed. Treatment with rituximab or VBP6 significantly decreased the dystrophic pathology score (Fig. 10). CTLA4-Ig, prednisolone and eplerenone did not show significant difference compared to AAV-microdystrophin treated mice. Active regeneration (basophilic fibers) was significantly increased in mdx compared to wild type but was not significantly altered by AAV-microdystrophin treatment with or without immunomodulatory drugs (Supplementary Figure 1, Supplementary Table 1).

Fig. 10

Dystrophic severity scores of H&E stained quadriceps muscles. Dystrophic severity was assessed with respect to the sample area that displayed active dystrophic disease, including myofiber degeneration, active myofiber regeneration (basophilic fibers), and inflammation. The area of each sample displaying these findings was visually estimated and the following grading scheme was applied: Grade 0 = normal, grade 1 = chronic regenerative changes only, grade 1.5 = very mild (< 5%of muscle area with active dystrophic pathology), grade 2 = mild (6–20%of muscle area with active dystrophic pathology), grade 2.5 = mild to moderate (21–30%of muscle area with active dystrophic pathology), grade 3 = moderate (30–50%of muscle area with active dystrophic pathology), grade 3.5 = moderate to severe (51–60%of muscle area with active dystrophic pathology) and grade 4 = severe (> 60%of muscle area with active dystrophic pathology). Areas indicative of chronic regeneration (internally nucleated fibers with appropriate eosinophilia, endomysial fibrosis, or fatty infiltration) were not integrated into the grading of dystrophic severity, as it is possible that the damage associated with these changes occurred before treatment. Data presented as mean or percentage±SEM. *P≤0.05, **P≤0.01 (upaired T-test). AAV- μDys AAV-microdystrophin, CT-Ig CTLA4-Ig, RTX rituximab, PRD prednisolone, EPL eplerenone.

Dystrophic severity scores of H&E stained quadriceps muscles. Dystrophic severity was assessed with respect to the sample area that displayed active dystrophic disease, including myofiber degeneration, active myofiber regeneration (basophilic fibers), and inflammation. The area of each sample displaying these findings was visually estimated and the following grading scheme was applied: Grade 0 = normal, grade 1 = chronic regenerative changes only, grade 1.5 = very mild (< 5%of muscle area with active dystrophic pathology), grade 2 = mild (6–20%of muscle area with active dystrophic pathology), grade 2.5 = mild to moderate (21–30%of muscle area with active dystrophic pathology), grade 3 = moderate (30–50%of muscle area with active dystrophic pathology), grade 3.5 = moderate to severe (51–60%of muscle area with active dystrophic pathology) and grade 4 = severe (> 60%of muscle area with active dystrophic pathology). Areas indicative of chronic regeneration (internally nucleated fibers with appropriate eosinophilia, endomysial fibrosis, or fatty infiltration) were not integrated into the grading of dystrophic severity, as it is possible that the damage associated with these changes occurred before treatment. Data presented as mean or percentage±SEM. *P≤0.05, **P≤0.01 (upaired T-test). AAV-
μDys AAV-microdystrophin, CT-Ig CTLA4-Ig, RTX rituximab, PRD prednisolone, EPL eplerenone.

DISCUSSION

In this study, we show that treatment with immunomodulatory (CTLA4-Ig, rituximab) and anti-inflammatory (prednisolone, VBP6 and eplerenone) drugs after AAV-microdystrophin gene therapy in mdx mice resulted in the following changes; a) body weight and tissue weights were reduced after gene therapy and these reductions were enhanced with prednisolone and VBP6, b) treatment with rituximab, prednisolone, VBP6 or eplerenone significantly reduced spleen weight, c) treatment with AAV-microdystrophin significantly increased the forelimb grip strength and isometric torque and these improvements were further increased with all immunomodulatory treatments with the exception of eplerenone, d) AAV-microdystrophin treatment significantly reduced cathepsin-B activity in both forelimbs and hindlimbs and addition of prednisolone and VBP6 further reduced cathepsin-B activity, e) fifty eight percent of AAV-microdystrophin treated animals showed anti-dystrophin antibodies and all immunomodulatory drugs significantly reduced the frequency of antibody positive animals with the exception of CTLA4-Ig, f) dystrophin specific T cell responses were present in one third of AAV-microdystrophin treated mice. Rituximab, prednisolone and VBP6 but not eplerenone and CTLA4-Ig reduced T cell responses (although this was not statistically significant), g) AAV-microdystrophin increased dystrophin levels to nearly 80%of normal and this was not significantly changed with immunomodulatory drugs and h) AAV-microdystrophin treated mice showed a lower dystrophic severity score and treatment with rituximab or VBP6 but not others further significantly decreased the dystrophic severity score.

In DMD, dystrophin deficiency leads to chronic membrane instability and self-sustaining activation of the innate immune response [21]. It is known that proinflammatory cytokines induce constitutive MHC class I and II expression on muscle cells, recruitment of T and B lymphocytes, and generation of an adaptive immune response in the muscle milieu [22–27]. This proinflammatory micro-environment is often superimposed on the recruitment of immune cells such as neutrophils, macrophages and dendritic cells induced by successive courses of myofiber degeneration and regeneration [28]. T-cells play an essential role in DMD pathogenesis [29, 30]. Blocking signal-2 and reduction of B lymphocytes to inhibit the immune response to autoantigens are widely used approaches to treat autoimmune disease conditions, such as rheumatoid arthritis. Using two FDA-approved biologics to block co-stimulation (CTLA4-Ig) or to reduce B cells (rituximab) to selectively block the dystrophin-specific immune and inflammatory response, could be used in DMD to block the immune response to gene therapy whilst avoiding the complications associated with traditional immunosuppressive drugs such as prednisolone.

In our study, mdx mice weighed more than age and sex-matched wild-type controls, consistent with the literature [31–33]. Increased body weight in mdx mice is caused by hypertrophy of the muscle [34], reflected in the high tissue weights of dissected muscle from mdx compared to wild type in this study. Treatment with AAV-microdystrophin reduced body weight gain in mdx mice towards wild-type levels. The addition of immunomodulatory drugs did not affect weight gain except for a marginally significant reduction for VBP6 (p < 0.05) and a highly significant decrease for prednisolone (p < 0.0001). This finding is consistent with the literature on prednisolone that shows, in contrast to the weight gain seen in children with DMD due to increased appetite [35], weight loss in mdx mice is due to the catabolic activity of this drug in the muscle [33, 36].

Treatment with AAV-microdystrophin significantly increased spleen weight which may indicate an immune response to the treatment. The addition of rituximab, prednisolone, VBP6, or eplerenone reduced spleen weight, possibly partly due to suppression of the immune response to the AAV-microdystrophin treatment and/or injured muscle.

In line with previous studies, we showed that forelimb grip strength is reduced in mdx mice compared to wild type [31]. Treatment with AAV-microdystrophin improved forelimb grip strength, and the addition of immunomodulatory drugs further improved forelimb grip strength force, except with eplerenone which made no significant difference. Prednisolone has previously been found to improve grip strength force in mdx mice [33]. VBP6 improved forelimb strength, consistent with the results from a related compound, VBP15 [37], suggesting that these drugs have similar beneficial effects.

We propose that all of these immunomodulatory drugs improve muscle force via the reduction of inflammation, which reduces the level of muscle damage in these mice. Variations in the mechanism of action of these drugs may explain the differences in the magnitude of response. For instance, prednisolone and VBP6 target the NFκB pathway in all immune cells [38] and therefore had a more significant beneficial impact on muscle function than CTLA4-Ig, which targets T-cells [39]. Interestingly, rituximab targets CD20+ B-cells [40] but shows an improvement in grip strength force similar to prednisolone. We postulate that this beneficial effect, which is beyond what would be expected from B-cell depletion alone, may be due to an alternative target of rituximab, smpdl3b, which is present on dendritic cells, macrophages [41], and muscle cells themselves (unpublished data). Therefore, rituximab may work directly on the muscle and dampen the muscle-damaging immune response in these mice.

The percentages of CD4+ and CD8+ T-cells were increased in the peripheral blood of mdx mice compared to wild-type controls consistent with a previous study of T-cells in mdx mice [29]. Treatment with AAV-microdystrophin had no significant impact on the percentage of CD4+, CD8+, CD11b+ or CD20+ cells within the CD45+ population. However, the percentage of CD4+ T-cells was reduced by the addition of rituximab or prednisolone. The percentage of CD11b+ cells was increased by the addition of prednisolone, consistent with the known property of prednisolone marginalizing leukocytes and increasing leukocyte numbers in the peripheral blood [42]. The percentage of CD20+ B-cells was increased by the addition of CTLA4-Ig and reduced by prednisolone.

As expected, optical imaging showed that mdx mice had significantly increased muscle cathepsin-B activity compared to wild-type controls, suggesting increased inflammation and/or muscle regeneration consistent with the literature [15]. Treatment with AAV-microdystrophin significantly reduced cathepsin-B activity, and this was further reduced with the addition of prednisolone and VBP6 (the other immunomodulatory drugs did not significantly change the levels of cathepsin-B activity).

Normalized isometric torque was significantly increased in AAV-microdystrophin treated in comparison to untreated mdx mice. The addition of immunomodulatory drugs significantly improved normalized torque compared to AAV-microdystrophin alone, except eplerenone which did not substantially change isometric torque. The improvement in normalized isometric torque was highly significant for prednisolone, and VBP6 treated mice. These immunomodulatory drugs may improve muscle function by reducing inflammation of the muscle, as described for grip strength above.

Western blot analysis was performed to detect anti-dystrophin antibodies present in mouse serum. Two out of ten untreated mdx mice had anti-dystrophin antibodies (20%). This reactivity could be due to an immune response to revertant fibers expressing dystrophin in these mdx mice. As expected, treatment with AAV-microdystrophin increased the percentage of mice with anti-dystrophin antibodies (57.8%) due to the immune response to the introduction of microdystrophin protein. All immunomodulatory drugs decreased the percentage of mice with anti-dystrophin antibodies due to their immunosuppressive properties (although this reduction was not statistically significant for the CTLA4-Ig group). None of the mice had detectable anti-dystrophin antibodies in the rituximab group, consistent with the B-cell depletion activity of this drug [40]. It should be noted that rituximab only targets CD20+ B-cells and not the antibody producing plasma cells. In this model, depletion of B-cells shortly after human microdystrophin gene therapy likely prevented development of plasma cells producing anti- human dystrophin antibodies. However, if applied in DMD patients there may be some pre-existing plasma cells producing anti-dystrophin antibodies due to revertant fibers expressing dystrophin. VBP6 had zero, and prednisolone had just one mouse with detectable anti-dystrophin antibodies. The reduction of an anti-dystrophin-specific response with immunomodulatory drugs may help to enhance the beneficial effects of microdystrophin gene therapy.

The production of IFNγ or IL-2 was used to assess dystrophin-specific T-cell responses after stimulation with dystrophin peptides. Treatment with AAV-microdystrophin increased the percentage of mice showing a T-cell response. However, not all mice showed T-cell responses, consistent with our previous observation of anti-dystrophin antibody and T-cell responses after PMO mediated exon skipping treatment in dystrophin-deficient mdx mice [20]. None of the immunomodulatory drugs had a statistically significant impact on the percentage of mice with dystrophin-specific T-cell responses. However, the percentage was reduced in the rituximab, prednisolone, and VBP6 groups compared to the eplerenone and CTLA4-Ig groups, suggesting that suppression of T-cell responses alone is unlikely to abolish cell-mediated immunity after rAAV treatment altogether. Furthermore, it should be noted that CTLA4-Ig may block the negative co-stimulation CTLA-4/B7 interactions in addition to the CD28/B7 co-stimulation.

One possible limitation of this approach is that T cell responses may be weaker in this context due to the use of human peptides that may not be recognized by mouse MHC molecules. However, the presence of anti-dystrophin IgG antibodies in this cohort of mice demonstrates that T cell epitopes recognized by mice are distinct from human epitopes used in our study.

Dystrophin expression was assessed by mass spectrometry. The AAV-microdystrophin treatment restored dystrophin levels to 80%of normal wild-type mice. The expression of dystrophin protein was not significantly altered by treatment with any of the immunomodulatory drugs in this study. We hypothesize that to increase muscle transduction after AAV-microdystrophin, treatments would need to target the initial innate immune response in addition to the downstream immune mechanisms targeted in this study. For instance, once administered, a substantial portion of the AAV may be cleared from circulation by phagocytic cells. Inhibition of these initial innate immune responses could enhance muscle transduction and result in even higher levels of microdystrophin present in the muscle following gene therapy.

As expected, muscle damage was observed on H&E stained sections of mdx muscle but absent in wild type. The dystrophic severity score was significantly reduced by treatment with AAV-microdystrophin. The addition of rituximab or VBP6 further significantly reduced the dystrophic severity score. The other immunomodulatory drugs demonstrated the same trend of reduced dystrophic severity score, but this did not reach statistical significance. Based on the data, we propose that these immunomodulatory treatments may reduce muscle damage by reducing inflammation in the muscle and allowing regeneration of the muscle. Furthermore, as discussed previously, rituximab may have additional benefits by directly targeting smpdl3b on the muscle itself.

ACKNOWLEDGMENTS

Funded by Parent Project Muscular Dystrophy, Foundation to Eradicate Duchenne and Solid Bioscience Inc.

Kitipong Uaesoontrachoon provided training for grip strength, isometric torque and necropsy. Carl A. Morris and J. Patrick Gonzalez provided AAV vector.

CONFLICT OF INTEREST

K.J.B. is an employee of Solid Biosciences Inc.

M.W.L., research funding provided by Solid Biosciences, Audentes Therapeutics, Taysha Therapeutics, and Prothelia. Board member for Solid Biosciences and Audentes Therapeutics during the period when studies were performed. Consultant for Audentes Therapeutics, AGADA Biosciences, Encoded Therapeutics, Kate Therapeutics, Lacerta Therapeutics, Affinia Therapeutics, Modis Therapeutics, Dynacure, and Biomarin.

K.N. is a co-founder of ReveraGen BioPharma and AGADA Biosciences.

SUPPLEMENTARY MATERIAL

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