PMO-based let-7c site blocking oligonucleotide (SBO) mediated utrophin upregulation in mdx mice, a therapeutic approach for Duchenne muscular dystrophy (DMD)

Upregulation of utrophin, a dystrophin related protein, is considered a promising therapeutic approach for Duchenne muscular dystrophy (DMD). Utrophin expression is repressed at the post-transcriptional level by a set of miRNAs, among which let-7c is evolutionarily highly conserved. We designed PMO-based SBOs complementary to the let-7c binding site in UTRN 3′UTR, with the goal of inhibiting let-7c interaction with UTRN mRNA and thus upregulating utrophin. We used the C2C12UTRN5′luc3′ reporter cell line in which the 5′- and 3′-UTRs of human UTRN sequences flank luciferase, for reporter assays and the C2C12 cell line for utrophin western blots, to independently evaluate the site blocking efficiency of a series of let-7c PMOs in vitro. Treatment of one-month old mdx mice with the most effective let-7c PMO (i.e. S56) resulted in ca. two-fold higher utrophin protein expression in skeletal muscles and the improvement in dystrophic pathophysiology in mdx mice, in vivo. In summary, we show that PMO-based let-7c SBO has potential applicability for upregulating utrophin expression as a therapeutic approach for DMD.


Results
Let-7c PMO SBO treatment showed increased utrophin expression in C2C12 cells. To determine the most efficient SBOs for achieving utrophin upregulation, we designed five different let-7c PMO SBOs (S24, S28, S31, S32 and S56) spanning different regions of the let-7c target site in the UTRN 3′UTR (Fig. 2a, Supplementary Table 1). To evaluate the efficacy of these PMOs, we used the C2C12UTRN5′luc3′ reporter cell line 39 containing the luciferase gene flanked by UTRN 5′ and 3′ UTRs for reporter assays. Four different concentrations for each PMO, were tested in the reporter cell line. S24 and S31 PMO treated cells showed ~ 1.5-fold higher luciferase activity compared to control PMO treated cells at 0.5 µM and 1 µM concentration respectively (Fig. 2b). The higher activity of S24 and S31 PMO was noted in a narrow range of concentrations. However, S56 PMO, which was designed based on the human UTRN let-7c sequence, showed ~ 1.3-fold increase in luciferase activity from 0.5 µM to 3 µM concentration, implying effectiveness over a wider range of active concentrations. Also the S32 PMO showed ~ 1.4-fold increase in luciferase activity from 0.5 µM to 3 µM concentration (Fig. 2b, Supplementary Table 3).
We further tested if the five PMOs could upregulate endogenous utrophin protein levels in C2C12 cells by using western blotting as an independent, orthogonal assay. Consistent with the results obtained with the reporter cell line, S56 PMO showed the highest level of utrophin expression (~ two-fold upregulation) compared to cells treated with control PMOs. Whereas the S32 PMO showed ~ 1.9-fold higher utrophin expression at 3 µM but not at other concentrations (Fig. 2c,d, Supplementary Table 4). Based on these findings S56 PMO was chosen for an in vivo preclinical study in the mdx mouse model of DMD. Mdx mice have a premature stop codon mutation in exon 23 of the DMD gene, and are widely used for preclinical animal studies for DMD 40,41 . Systemic administration of S56 PMO upregulates utrophin in mdx mice. PK/pharmacodynamic (PD) studies of PMOs have shown highest bioavailability upon intravenous (i.v) administration 35 . We therefore treated five weeks old mdx mice with five weekly retro-orbital injections of S56 PMO or control PMO at a dosage of 80 mg/kg. Two weeks after the end of the treatment, mice were sacrificed, and their skeletal muscles  www.nature.com/scientificreports/ were examined (Fig. 3a). Western blots showed significant increases in utrophin protein expression in gastrocnemius (~ 1.6-fold) and soleus (~ 2.5-fold) muscles treated with S56 PMO compared to control PMO treated mice (Fig. 3b,c). Surveyed muscles from S56 and control PMO treated mice showed higher utrophin protein and mRNA expression levels, consistent with the intervention (Supplementary Figs. 1-2). We further tested for expression of β-dystroglycan, which is a hallmark for restoration of dystrophin-glycoprotein complex (DGC). Western blots showed partial restoration of β-DG in S56 PMO treated TA and EDL muscles ( Supplementary Fig. 3). S56 PMO treatment increased sarcolemmal utrophin expression in mdx mice. As the western blots showed higher expression of utrophin protein in S56 PMO treated mdx mice, we examined utrophin protein expression in the sarcolemma of control and S56 PMO treated mdx mice by immunohistochemistry. Control PMO treated TA sections showed utrophin expression primarily restricted to the neuromuscular junctions, as indicated by the colocalization with the synaptic marker α-Bungarotoxin (BTX). On the other hand, muscles from mice treated with S56 PMO showed increased expression of utrophin across both synaptic and extrasynaptic regions (Fig. 4a). Quantification of utrophin level normalized to wheat germ agglutinin (WGA) levels in sarcolemma showed ~ two-fold higher expression of utrophin in TA muscles of S56 PMO treated mdx mice (Fig. 4b).
S56 PMO treated mdx mice showed decrease in muscle degeneration and morphological improvement. We further examined whether the dystrophic histopathology was improved by S56 PMO treatment compared to control PMO treatment in mdx mice. H&E staining of EDL and diaphragm sections of control PMO treated mdx mice showed regenerating centrally nucleated fibers (CNFs) and immune cell infiltration as previously reported 40 . However, let-7c PMO treatment of age-matched mdx mice showed improvement in the pathophysiology, with reduced CNFs, necrosis and cellular infiltration observable in EDL and diaphragm ( Fig. 5a-c). The higher percentage of CNFs is one of the well-recognized, characterized and objectively scored morphological characteristics of dystrophic muscles, attributed to continuous muscle regeneration 40,43 . Two weeks after the end of treatment, EDL and diaphragm muscles of mdx mice showed significantly lower percentage of CNFs in S56 PMO treated mice (c.a. 13% reduction) compared to control PMO treated mice, providing morphological evidence of improvement of the dystrophic phenotype (Fig. 5d,e). The morphological improve-  Table 2).

S56 PMO treatment decreases serum CK levels in mdx mice.
We further studied the therapeutic effects of the let-7c PMO treatment on mdx mice by examining the levels of serum CK. Elevated CK level is one of the hallmarks of dystrophic pathophysiology in mice and humans 40,42 . Two weeks after the end of the treatment, serum CK was significantly lower (c.a. 38% reduction) in S56 PMO treated compared to control PMO treated mdx mice (Fig. 5f). The decrease in serum CK provides biochemical evidence of utrophin mediated improvement in dystrophic pathology.

Discussion
In this study, we developed and used the S56 PMO-based let-7c SBO to alleviate miRNA mediated repression of utrophin expression and achieved improvement in mdx pathophysiology in vivo. Utrophin mRNA translation is known to be inhibited by a set of six miRNAs 25,26 . Bulaklak et al., showed AAV mediated expression of a miR-206 sponge decoy 44,45 in mdx mice, resulted in utrophin upregulation and improvement in the dystrophic phenotype, independently demonstrating the importance of miRNA mediated regulation of utrophin expression 27 . We had shown higher utrophin expression and improvement of the dystrophic phenotype by blocking the let-7c target site in UTRN 3′UTR using a 2OMePS-based let-7c SBO in vivo 30 . Additionally, sPIF-mediated let-7 downregulation has also been shown to result in utrophin upregulation and improvement in the dystrophic phenotype 46 , underscoring the significance of let-7 mediated regulation of utrophin expression. However, the clinical application of 2OMePS-based let-7c SBO for utrophin upregulation or 2OMePS-based DMD exon skipping 47 AONs is somewhat compromised by their suboptimal PKs and therapeutic efficacy 34 . Whereas, PMOs while clinically approved by the FDA, have sub-optimal efficacy 36,37,48 . PK studies of PMOs show rapid elimination from bloodstream and limited entry to mature muscle fibers 49 . However, a recent study by Novak et. al. demonstrated presence of DMD exon skipping PMOs in macrophages at the site of myofiber lesions for several days after elimination of PMOs from bloodstream, demonstrating the availability of PMOs at actively regenerating myofibers 50,51 . Here, we tested the efficacy of five different let-7c PMO-based SBOs for utrophin upregulation in vitro and studied the most efficient PMO (S56 PMO) in mdx mice to demonstrate improvement in dystrophic pathophysiology. AAV mediated μ-utrophin delivery [52][53][54] and artificial transcriptional factor mediated transcriptional upregulation of full length utrophin 24,55 respectively, have been shown to improve dystrophic pathophysiology in the mdx model. Notably, μ-utrophin expression does not induce significant immune responses compared to μ-dystrophin expression in deletional animal models of DMD 54 . In this study, weekly S56 PMO administration for 5 weeks resulted in ~ 2.5-fold and ~ 1.6-fold higher level of utrophin in soleus and gastrocnemius muscle respectively, www.nature.com/scientificreports/ biochemical and morphological improvement as demonstrated by a 38% reduction in serum CK levels and ~ 13% reduction in percentage of CNFs compared to control PMO treated mdx mice (Figs. 3, 4, 5). While improvement in these parameters are extremely encouraging, the improvement achieved at this dose was limited (Supplementary Table 2). Our previous studies using two different doses (10 mg/kg and 100 mg/kg) of 2OMePS let-7c SBO showed dose-dependent utrophin protein upregulation in skeletal and diaphragm muscle and improvement in mechanical properties only at the higher dose 30 . However, even at the higher dose of 2OMePS let-7c SBO, the serum CK level showed no improvement. Here, with a relatively low dose of 80 mg/kg S56 PMO treatment for five weeks in mdx mice showed increased expression of utrophin, morphological improvement and a decrease in serum CK level. We were unable to test higher doses of our lead PMO-based let-7c SBO due to volume constraints of retrobulbar delivery, coupled with the high GC content of the target site, which limited the solubility of the S56 PMO SBO we developed. We believe that designing and testing of additional SBOs and/or use of alternate   56,57 , and we are currently undertaking these experiments to increase the potential therapeutic efficacy. We suggest that the functional improvement we achieved can be further improved with an earlier onset of treatment, a longer period of treatment and/or administration of higher PMO dosages. Given that PMOs have demonstrated sufficient safety and efficacy in vivo at higher doses (100-300 mg/kg/wk) 48,58-60 , the 80 mg/kg/wk we used should not be limiting, however, additional sequences based on S56 would need to tested to quantify dose-dependent improvements. Additionally, here we have targeted one specific miRNA::utrophin interaction, however this approach can easily be extended to additional miRNAs that we and others have identified 25,26 singly (f) The graph shows serum CK levels in S56 PMO treated mice were significantly lower than control PMO treated mdx mice (*p = 0.02, n = 10 mice for both groups). Each bar represents mean ± SEM. Statistical analysis performed by Mann-Whitney nonparametric test.

PMO-based SBO design.
The PMO backbone let-7c SBOs were designed to specifically target and block the let-7c miRNA target site in UTRN 3′UTR. Five let-7c PMOs were designed targeting the same let-7c site but with different flanking regions considering the binding efficiency. A control PMO was designed with the scramble sequence. All the PMOs were synthesized by Gene Tools, LLC (OR) (Supplementary Table 1).
Luciferase assay. The C2C12UTRN5′luc3′ cell line was seeded at 50,000 cells/well in 24 well plates and allowed to attach for O/N as described 39 . Next day, cells were treated for 24hrs. with control or let-7c PMOs of desired concentrations (0.1 µM, 0.5 µM, 1 µM, 3 µM) along with 6 µM transfection reagent endoporter (Gene Tools, LLC, OR). The next day, treated cells were lysed using the passive lysis buffer (Promega, WI) supplemented with complete protease inhibitor (Roche, Switzerland) and total protein contents were measured with the Pierce BCA protein assay kit (Thermo Fisher Scientific, MA). Luciferase assays were done using the luciferase assay system (Promega, WI) in a 96 well plate and quantified using a Cytation5 plate reader (BioTek, VT). Luciferase activity was normalized to the total protein loaded.
Animal studies. All animal procedures were reviewed and approved by the Institutional Animal Care and Preclinical studies in mdx mice with PMOs. PMOs were solubilized in saline and warmed at 50 °C for 15mins and cooled down to room temperature before injections. Five-week-old male mdx mice were anesthetized with 4% isoflurane and control or S56 PMO was administered systemically via the retro-orbital sinus as a single 80 mg/kg dose (< 250 µl volume) using a 28-gauge/0.5 ml insulin syringe, weekly for 5 weeks. 2 weeks after the last injection, mice were anesthetized by 4% isoflurane and sacrificed. Ex vivo contractility analyses were performed as previously described 30 . Following the procedure, muscles were flash-frozen in liquid nitrogen-cooled isopentane and stored at − 80 °C. www.nature.com/scientificreports/ Reprints and permissions information is available at www.nature.com/reprints.
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