Genome Editing-Mediated Utrophin Upregulation in Duchenne Muscular Dystrophy Stem Cells

Abstract

Utrophin upregulation is considered a promising therapeutic strategy for Duchenne muscular dystrophy (DMD). A number of microRNAs (miRNAs) post-transcriptionally regulate utrophin expression by binding their cognate sites in the 3′ UTR. Previously we have shown that miRNA: UTRN repression can be alleviated using miRNA let-7c site blocking oligonucleotides (SBOs) to achieve utrophin upregulation and functional improvement in mdx mice. Here, we used CRISPR/Cas9-mediated genome editing to delete five miRNA binding sites (miR-150, miR-296-5p, miR-133b, let-7c, miR-196b) clustered in a 500 bp inhibitory miRNA target region (IMTR) within the UTRN 3′ UTR, for achieving higher expression of endogenous utrophin. Deleting the UTRN IMTR in DMD patient-derived human induced pluripotent stem cells (DMD-hiPSCs) resulted in ca. 2-fold higher levels of utrophin protein. Differentiation of the UTRN edited DMD-hiPSCs (UTRNΔIMTR) by MyoD overexpression resulted in increased sarcolemmal α-sarcoglycan staining consistent with improved dystrophin glycoprotein complex (DGC) restoration. These results demonstrate that CRISPR/Cas9-based UTRN genome editing offers a novel utrophin upregulation therapeutic strategy applicable to all DMD patients, irrespective of the dystrophin mutation status.

Graphical Abstract

Sengupta et al. report a genome editing strategy for upregulating utrophin in Duchenne muscular dystrophy patient iPSCs. Edited iPSC-derived myotubes demonstrate increased expression of utrophin and components of the dystroglycan/sarcoglycan complex. This approach represents a promising therapeutic strategy applicable for all DMD patients, irrespective of their mutation status.

Introduction

Duchenne muscular dystrophy (DMD) is an X-linked recessive disease affecting approximately 1 in ∼5,000 live born males worldwide.^1,2 DMD is caused by mutations in the DMD gene, resulting in the loss or extremely low expression of the dystrophin protein.^3,4 Dystrophin is a 427 kDa cytoskeleton-associated protein and member of dystrophin glycoprotein complex (DGC), which functions as a linker between the extracellular matrix and the intracellular actin.5, 6, 7 Dystrophin provides structural integrity to myofibers during cycles of contraction and relaxation.⁸ In the absence of dystrophin, the increased sarcolemmal fragility leads to repeated cycles of muscle degeneration and regeneration, which inexorably result in replacement of contractile tissue with fibrotic and adipose tissues.^9,10 DMD patients typically present by school age and a loss of ambulation by their teens. The disease continues to progress leading to increasing degrees of skeletal muscle weakness and respiratory and cardiac failure, typically by the fourth decade of life.¹¹

Different genetic and pharmacological interventions designed to restore dystrophin expression by antisense oligonucleotide-mediated exon skipping,12, 13, 14 stop codon readthrough,¹⁵ dystrophin gene delivery,16, 17, 18 and CRISPR/Cas9 genome editing either to restore an open reading frame or to delete mutated exons19, 20, 21, 22 are currently in different stages of preclinical or clinical studies. However, there are numerous challenges associated with toxicity, the necessity for systemic delivery of these approaches, as well as, the lack of global applicability to patients due to the existence of a variety of dystrophin mutations that cause DMD (https://www.dmd.nl/database.html).

A promising therapeutic approach for DMD that circumvents many of these issues is upregulating the dystrophin-related protein, utrophin: the chromosome 6-encoded autosomal paralog of dystrophin, with a high degree of structural and functional similarity to dystrophin.23, 24, 25 The major utrophin isoform in myofibers utrophin-A, is enriched in neuromuscular and myotendinous junctions of adult muscles and at the sarcolemma of regenerating myofibers.26, 27, 28, 29 Small molecules such as heregulin,³⁰ nabumetone,³¹ SMT C1100,³² and artificial transcription factors^33,34 have been shown to upregulate UTRN gene expression by activating the UTRN-A promoter. In addition, utrophin can be upregulated by peptides and redistributed by biglycan-mediated protein anchoring at the post-transcriptional level.35, 36, 37 Previously, we and others have shown the existence of post-transcriptional inhibition of utrophin expression by a number of miRNAs targeting UTRN 3′ UTR.^38,39 Suppressing one of those miRNA interactions with site blocking oligonucleotides (SBOs) for let-7c resulted in the utrophin upregulation-mediated functional improvement in mdx mouse.⁴⁰ While providing a proof of concept, this approach is limited by the far from ideal pharmacological properties of SBO’s in vivo.⁴¹

In this study, we describe a CRISPR/Cas9-based utrophin genome editing strategy designed to upregulate utrophin expression by targeting post-transcriptional miRNA-mediated inhibition for DMD (Figure 1). In the native (unedited) state, the five miRNA binding sites (for miR-150, miR-296-5p, miR-133b, miR-196b, and let-7c) that are clustered at the inhibitory miRNA target region (IMTR) repress utrophin expression. We propose that CRISPR/Cas9 genome editing-mediated deletion of the UTRN-IMTR would eliminate the miRNA-binding sites at this locale, leading to increased utrophin expression and results in amelioration of the dystrophic phenotype in DMD, in vivo.

Figure 1.

CRISPR/Cas9 Strategy for Relieving miRNA Driven Post Transcriptional Repression and Increased Expression of Utrophin

The schematic summarizes our CRISPR/Cas9 genome editing strategy to delete the IMTR from the UTRN 3′ UTR with the rationale that the edited UTRN 3′ UTR (UTRNΔIMTR) would reduce miRNA-mediated post-transcriptional repression and lead to higher expression of utrophin.

Results

CRISPR/Cas9 Genome Editing Strategy to Delete the IMTR of UTRN

We have previously shown that UTRN gene expression is regulated by five inhibitory miRNAs targeting the 3′ UTR³⁸ (Figure 2A). To delete these miRNA-binding sites clustered in the IMTR of the UTRN 3′ UTR, we designed four compatible short guide RNAs (sgRNAs 1–4; Table S1) targeting the flanking region of IMTR in the human UTRN gene. Both SaCas9 and the sgRNA pairs were cloned in the same vector (p-UTRNΔIMTR) and transfected in Human embryonic kidney 293T (HEK293T) cells to determine the deletion efficiency and validate the editing (Figure 2B). We used a genomic PCR screening strategy to detect successful deletion of the target region using a primer pair flanking the IMTR for PCR screening (Figure 2C). Gel electrophoresis of PCR products demonstrated that the sgRNA pairs 1 and 4 deleted IMTR most efficiently (Figure 2D; Figure S1). PCR products were also subjected to DNA sequencing to confirm precision of editing (data not shown), prior to utilizing this strategy in human induced pluripotent stem cells (hiPSCs).

Figure 2.

CRISPR/Cas9 Genome Editing Targeting UTRN 3′ UTR

(A) Schematic diagram of UTRN gene showing relative positions of five inhibitory miRNA target sites (miR-150, miR-296-5p, miR-133b, let-7c, and miR-196b) located in the 3′ UTR. The IMTR is shown as red block. The SaCas9 sgRNA target sites are designed flanking the IMTR shown as red arrows. (B) Scheme of HEK293T cell transfection with a plasmid construct (p-UTRNΔIMTR) containing SaCas9 and dual sgRNAs (sg) followed by genomic DNA isolation and PCR screening for UTRNΔIMTR. (C) Scheme showing PCR screening strategy for identifying sgRNA pairs to efficiently achieve UTRNΔIMTR editing. The red lightning bolts show SaCas9 cut sites. (D) The DNA gel shows genomic PCR analysis from HEK293T cells transfected with different combinations of sgRNA pairs. The larger PCR products (800 bp) are from unedited UTRN and shorter PCR products (250–350 bp) are from UTRNΔIMTR gene.

Genome Edited UTRNΔIMTR DMD-hiPSC Lines Show Utrophin Protein Upregulation

A DMD patient fibroblast-derived hiPSC cell line²² carrying a deletion of DMD exons 46–51 (DMD-hiPSC) was subjected to sgRNA 1 and 4 pair directed CRISPR-Cas9-mediated genome editing followed by fluorescence-activated cell sorting (FACS) isolation of transfection positive population and clonal selection (Figures 3A and 3B). Stably deleted DMD-hiPSC line clones were screened for homozygous IMTR deletion (UTRNΔIMTR) using the PCR strategy described above (Figure 3C). Deletions were confirmed by sequencing PCR products from the edited clones (Figure 3D). Utrophin protein expression in UTRNΔIMTR and sham-edited DMD-hiPSC were compared by western blotting and UTRNΔIMTR clones showed up-to 2-fold utrophin upregulation (Figures 3E and 3F).

Figure 3.

UTRNΔIMTR Genome Editing in DMD-hiPSCs and Utrophin Protein Upregulation in UTRNΔIMTR Clones

(A) Fluorescence and bright-field microscopy images showing GFP expression in DMD-hiPSCs transfected with SaCas9-GFP/sgRNA1 and 4. Scale bar, 200 μm. (B) FACS sorting of GFP-positive DMD-hiPSCs gated against mock transfected DMD-hiPSCs. (C) Genomic DNA PCR gel from clonally selected, genome edited, DMD-hiPSC lines shows a 267 bp band from UTRNΔIMTR gene (UTRNΔIMTR clones a, b, and c). Whereas genomic DNA PCR from the sham edited (cells transfected with only SaCa9 and no sgRNA) cells shows only unedited 800 bp band. (D) DNA Sequencing of PCR product from UTRNΔIMTR clone shows precise (533 bp) deletion of IMTR compared to sham edited clone. The W in the chromatogram stands for T/A. (E) Representative western blot shows expression of utrophin in DMD-hiPSC sham edited and UTRNΔIMTR clones. α-tubulin was used as loading control. (F) Densitometric analysis of utrophin western blot to quantify utrophin upregulation. Bands were densitometrically quantified and utrophin normalized to α-tubulin. Error bars represent mean ± SEM (n = 4). Difference in utrophin expression between clones were statistically analyzed by the Mann-Whitney test (∗p ≤ 0.05). Significant increase in utrophin expression was observed in UTRNΔIMTR clones 2, 3, and 5 compared to sham edited clones with p value 0.028.

Validation of hiPSC Clones Post-genome Editing

Pluripotency of the wild-type, DMD, and edited UTRNΔIMTR hiPSC clones was confirmed by immunostaining for nuclear expression of the pluripotency marker Nanog⁴² (Figure 4). The top five potent off-target sites of the sgRNA 1 and 4 used for genome editing were determined with the COSMID bioinformatics-based tool⁴³ (Tables S2 and S3) and sequenced by PCR amplification of the loci. No off-target mutations were observed at these sites in the selected UTRNΔIMTR clones demonstrating the precise nature of genome editing of the guide RNA pairs.

Figure 4.

Pluripotency Marker Nanog Expression in Wild-Type, DMD, and UTRNΔIMTR hiPSC Clones by Immunostaining

Immunofluorescent staining images of wild-type, DMD, and UTRNΔIMTR hiPSC clones with DAPI (blue), Nanog (red), and α-tubulin (green) for validation of pluripotency in post-genome edited lines. The merge panel at bottom shows nuclear localization of pluripotency marker Nanog in different clones. Magnification 20×, scale bar, 200 μm.

Characterization of hiPSCs Differentiated to Myogenic Lineage by MyoD Overexpression

The wild-type, DMD, and selected UTRNΔIMTR hiPSC clones were differentiated to myogenic lineage using a tamoxifen inducible MyoD expressing lentivirus²² (Figure 5A). The edited and unedited fused, multinucleated myotubes showed positive myosin heavy chain (MYHC) expression by immunostaining upon differentiation (Figure 5B). For all the three differentiated lines, 40%–50% of multinucleated myotubes were MYHC-positive myotubes (Figure 5C). Expression of the myogenic genes (MyoD1, MyoG [Myogenin], and endogenous MyoD1), as well as the pluripotency marker Nanog, were quantified in the UTRNΔIMTR cells by quantitative PCR (qPCR) at day 0, day 4, and day 8 post-tamoxifen induction. The qPCR profile showed a sharp decline in Nanog expression and a concomitant increase in MyoD1, MyoG, and endogenous MyoD1 genes, supporting differentiation of the UTRNΔIMTR cells to a myogenic lineage (Figure 5D). Utrophin expression in the differentiated myotubes were quantified by western blotting. UTRNΔIMTR myotubes showed higher utrophin expression compared to DMD myotubes (Figure S4).

Figure 5.

MyoD-Mediated Directed Differentiation of hiPSC Clones to Myogenic Lineage

(A) Schematic of myogenic differentiation of hiPSCs achieved by lentivirus mediated MyoD overexpression. (B) Differentiated wild-type, DMD, and UTRNΔIMTR myotubes were stained with DAPI (blue) and MYHC (green). Scale bar, 200 μm (C) Efficiency of myogenic differentiation determined by the fusion index (percentage of MYHC-positive myotubes with more than 2 nuclei). We counted a total of 85, 98, and 80 myotubes of wild-type, DMD, and UTRNΔIMTR, respectively. Average from three wells (three random fields from each well) with ± SEM are shown. (D) Gene-expression analysis by qPCR of MyoD infected UTRNΔIMTR clone in tamoxifen untreated (day 0) and tamoxifen treated (day 4 and day 8) point. Expression of pluripotency marker NANOG, skeletal muscle marker MyoD1, MyoG, and endogenous MyoD1 are shown (n = 3).

Utrophin Overexpression Increases Sarcolemmal α-Sarcoglycan Expression in UTRNΔIMTR hiPSC-Derived Myotubes

The absence of dystrophin protein in DMD muscles results in the disruption of the DGC and the lack of sarcolemmal staining for different components of the DGC, such as α-sarcoglycan.^44,45 Restoration of individual DGC proteins expression at the sarcolemma suggests restoration of the DGC and considered a marker of improvement when evaluating dystrophin or utrophin-based therapeutic strategies.46, 47, 48, 49 We therefore tested whether upregulated utrophin could increase sarcolemmal α-sarcoglycan expression in UTRNΔIMTR DMD-hiPSC-derived myotubes, by immunostaining. The UTRNΔIMTR DMD-hiPSC-derived myotubes showed significantly higher α-sarcoglycan level compared to the DMD-hiPSC-derived myotubes, supporting the restoration of utrophin anchored DGC by genome editing in the DMD-hiPSCs (Figures 6A and 6B). The increases noted on immunostaining was independently supported by western blot data showing overall higher expression of α-sarcoglycan in UTRNΔIMTR DMD-hiPSC-derived myotubes compared with DMD-hiPSC-derived myotubes (Figure 6C). Consistent with the utrophin upregulation mediated restoration of the DGC, we showed restoration of another DGC member, β-dystroglycan in UTRNΔIMTR DMD-hiPSC-derived myotubes by immunostaining (Figure S2).

Figure 6.

α-Sarcoglycan Expression in Differentiated Myotubes

(A) Wild-type, DMD, and UTRNΔIMTR differentiated myotubes were stained with DAPI (blue), MYHC (green), and α-sarcoglycan (red). Scale bar, 200 μm. (B) α-sarcoglycan quantification in wild-type, DMD, and UTRNΔIMTR differentiated myotubes. The α-sarcoglycan intensity shown as percentage expression mean ± SEM, was calculated as median of line intensity profile (n = 20) in ImageJ and normalized with DAPI expression. Differences in α-sarcoglycan expression between individual groups were analyzed by the Kruskal-Wallis multiple comparison test. The p value is <0.0001. (C) The western blot shows α-sarcoglycan expression in wild-type, DMD, and UTRNΔIMTR myotubes. Vinculin was used as loading control.

Discussion

The rapid developments in genome editing has generated enormous excitement and hope for treating devastating diseases such as DMD.50, 51, 52, 53 In this study, we describe a CRISPR/Cas9-mediated genome editing approach for increasing utrophin expression, as a therapeutic strategy for DMD (Figure 1). We used this approach to delete a 500 bp IMTR containing five miRNA binding sites (i.e., miR-150, miR-296-5p, miR-133b, let-7c, and miR-196b) within the UTRN 3′ UTR in HEK293T cells and select appropriate sgRNAs pairs (Figure 2). To test the strategy, we used sgRNA pairs 1 and 4 to delete the IMTR from DMD-hiPSCs (Figures 3A and 3D) and validated the UTRNΔIMTR DMD-hiPSC clonal lines for utrophin upregulation by western blotting (Figures 3E and 3F) and expression of the pluripotency marker Nanog by immunofluorescence (Figure 4). Lentivirus driven MyoD-mediated myogenic differentiation was utilized to drive the hiPSCs into myotubes and differentiation validated by monitoring the fusion index, as well as reduced expression of pluripotency marker and increased levels of myogenic markers by qPCR (Figure 5). Upon differentiation to myotubes, higher α-sarcoglycan levels were noted in edited compared to unedited DMD myotubes (Figure 6), suggestive of functional improvement due the UTRN genome editing, we describe in this study.

Previously described CRISPR/Cas9-mediated genome-editing-based therapeutic strategies for DMD have largely focused on editing dystrophin and met with varying degrees of success in preclinical studies.19, 20, 21, 22^,50,51,54 In common, these approaches, while extremely encouraging in preclinical studies, have fundamental limitations in that they would not be applicable to all DMD patients, need to be custom-designed for specific mutations, and would be predicted to be limited by immunity to the newly expressed dystrophin.⁵⁵ Nevertheless, adeno-associated virus (AAV)-mediated CRISPR genome editing in larger animal model of DMD to correct the dystrophin mutation and express a shorter form of dystrophin supports the efficacy and promise of using genome editing for DMD.⁵⁶ Dystrophin-independent CRISPR/Cas9 editing approaches have also been described for leveraging myostatin⁵⁷ and transcriptional activation of utrophin^58,59 as potentially therapeutic approaches. Our approach targets post-transcriptional mechanisms for increasing utrophin expression by deleting the miRNA target sites located in the IMTR of the UTRN 3′ UTR. The advantages of our approach are that other cellular targets of respective miRNAs remain unperturbed, this single editing strategy is applicable to all DMD patients, and a predicted lack of immune issues since DMD patient are not utrophin naive as they express utrophin since before birth.²⁴

In vivo preclinical studies using these targets leveraged by genome editing have been achieved using iPSCs, as well as AAV-mediated editing with varying degrees of success. AAV-based approaches have the advantage of enabling the same therapeutic viral vector(s) to be used in a number of patients and ease of delivery. However, the AAV-based approaches have limitations related to the cloning capacity, long-term expression of Cas9, and immune reactions against the capsid or cargo (i.e., Cas9,⁵⁴ Dsytrophin⁵⁵). While our study was restricted to genome editing of iPSCs in vitro, editing of autologous and/or allogenic iPSCs coupled with transplantation is a promising approach that has been used in vivo in a variety of disease models including DMD.^22,52,60, 61, 62 Both approaches have limitations but major efforts are ongoing to harness the potential of these approaches. Indeed, the recent demonstration that AAV9-mediated editing can transduce muscle satellite cells⁶³ and stem cells⁶⁴ exemplify the rapid pace of progress toward applying these strategies to develop therapies in DMD. Additionally, the UTRN genome editing strategy and proof-of-principle studies described here could potentially be combined with full-length utrophin, miniaturized utrophin (μUtro) upregulation,^55,65 or utrophin-independent approaches for synergistic effects.

Materials and Methods

Cell Culture and Maintenance

HEK293T cells (ATCC) were maintained in standard growth condition in DMEM high glucose (GIBCO) supplemented with 10% fetal bovine serum (Sigma-Aldrich) and 1% Pen/Strep (GIBCO).

All hiPSCs were reprogrammed from skin fibroblast with STEMCCA cassette in Dr. April Pyle’s laboratory, UCLA, as described in Karumbayaram et al.⁶⁶ We used two different hiPSC lines, one derived from a healthy individual (Wt 1002) and the other one derived from DMD patient harboring exon 46–51 deletion (CDMD1003). All the hiPSCs were grown in hESC-qualified Matrigel (Corning), fed daily with mTeSR 1 media (STEMCELL Technologies) as previously described and passaged every 4–5 days.

sgRNA Design and Cloning

All guide RNAs for generating the IMTR deletion (UTRNΔIMTR) were designed using the Benchling web tool for CRISPR design (Table S1). The CMV promoter of pX601 plasmid (Addgene plasmid # 61591) was replaced with EF1α promoter for improved expression of SaCas9 and an EGFP cassette was cloned at C-terminal of SaCas9 (pX601-EF1α::SaCas9-GFP). Individual sgRNA oligonucleotides were annealed and cloned in this modified pX601 plasmid at the BsaI restriction site before the sgRNA scaffold according to the protocols from the Zhang lab (https://www.addgene.org/crispr/zhang/; pX601-EF1α::SaCas9-GFP-U6::sgRNA). For expression of dual sgRNAs in the same plasmid, the second sgRNA under U6 promoter, were PCR amplified from the corresponding plasmid and subcloned at KpnI site of pX601-EF1α::SaCas9-GFP-U6::sgRNA. The cloned plasmids were confirmed by sequencing (Figure S3).

Genome Editing Validation of sgRNAs

HEK293T cells were transfected with plasmids containing SaCas9 and different pairs of sgRNAs using Lipofectamine 3000 (Invitrogen). The cells were suspended in DirectPCR Lysis Reagent (Viagen Biotech) and incubated with proteinase K for 6 hr at 55°C and heat inactivated at 85°C for 45 mins. 1 μL of genomic DNA (gDNA) extract were directly used for PCR screening of UTRN-IMTR deletion. In brief, a UTRN forward primer (5′-CCTTTCGGGTGAAAGATCAG-3′) and UTRN reverse primer (5′-ACTTACTTCCCATTGTTACTGC-3′) were used to amplify a fragment spanning the IMTR with GoTaq Green Master Mix (Promega), using the following cycling conditions: 95°C for 5 mins, 34 cycles at 95°C for 30 s, 60°C for 30 s, 72°C for 1 min, and final extension at 72°C for 10 mins. The PCR products and TrackIt 100 bp DNA ladder (Thermo Fisher Scientific) were electrophoresed on a 2% agarose gel. Gel images were captured using a G:Box imaging system (SYNGENE).

Electroporation of hiPSC Lines

Approximately 5 × 10⁶ hiPSCs were harvested using Accutase solution (Sigma-Aldrich) and washed in phosphate buffered saline (PBS, without Ca⁺² and Mg⁺²). Harvested cells were suspended in 75 μL of Resuspension Buffer R (Neon Kit, Invitrogen) and mixed with 25 μg of plasmid DNA. Cells were electroporated with three 10 ms pulses at 1,200 V (Neon Transfection System, Thermo Fisher Scientific). Post-electroporation cells were plated on Matrigel coated plates in mTeSR 1 media with 5 μM ROCK inhibitor (Y-27632, STEMCELL Technologies).

FACS of hiPSC Lines

48 h post-electroporation GFP-positive hiPSCs were FACS sorted in BD FACS Jazz System (BD Biosciences) at the FACS core of The Children Hospital of Philadelphia. Cells were harvested and suspended as single cells in FACS buffer (PBS, 1% FBS, 1 mM EDTA, 5 μM Y-27632). GFP-positive cells were gated with reference to mock electroporated GFP-negative cell population. FACS sorted GFP-positive hiPSCs were plated immediately in pre-warmed Matrigel coated 10 cm plate (5,000–10,000 cells/10 cm plate) with mTeSR™ 1 media supplemented with 10% CloneR (STEMCELL Technologies).

UTRNΔIMTR hiPSC Colony Screening

FACS sorted hiPSCs formed visible colonies by 7 days in culture. Colonies were picked and split in 96 well Matrigel coated plate with mTeSR™ 1 media. After 3 days cells were split and half harvested for genomic DNA extraction with DirectPCR Lysis Reagent (Viagen Biotech). The gDNA was used for PCR screening of UTRN-IMTR deletion with the primer pairs flanking UTRN-IMTR, as mentioned above. Positive homozygous colonies were selected for further expansion.

Western Blot

Cells were lysed in radioimmunoprecipitation assay (RIPA) buffer (20 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% NP-40, 1% sodium deoxycholate, 2.5 mM sodium pyrophoshphate, 1 mM β-glycerophoshphate, 1 mM sodium orthovanadate) supplemented with complete protease inhibitor cocktail (Roche). Total protein was measured by Pierce BCA Protein assay kit (Thermo Fisher Scientific). 10 μg of total protein was resolved in 3%–8% Tris-Acetate protein gel (NuPAGE, Thermo Fisher Scientific) and transferred to nitrocellulose membrane using Trans-Blot Turbo Transfer System (Bio-Rad). For immunoblotting, membranes were first blocked with 5% non-fat dry milk in TBS with 1% Tween20 for 1 h in room temperature. After blocking, blots were incubated with the following primary antibodies; mouse monoclonal anti-utrophin (1:100, Mancho3[8A4], developed by Prof. Glenn E. Morris; DSHB, IA, USA) and mouse anti-α-tubulin (1:5,000, T6199, Sigma-Aldrich) for overnight at 4°C. Next day, blots were washed; incubated with mouse immunoglobulin Gκ (IgGκ) binding protein (m-IgGκ BP) conjugated to horseradish peroxidase (HRP) (1:2,500, sc-516102, Santa Cruz Biotechnology); washed and developed using Immobilon Western Chemiluminescent HRP Substrate (Millipore) and imaged in LI-COR C-Digit Blot Scanner (LI-COR Biosciences-US).

Immunostaining

Cells were grown in Matrigel coated slide chambers, fixed with freshly prepared 4% paraformaldehyde (PFA) for 15 mins, permeabilized with 0.25% Triton X-100 for 5 mins, blocked with 4% BSA for 1 h, and stained with primary antibody overnight at 4°C. The following primary antibody dilutions were used: rabbit monoclonal anti-Sox2 (1:200, #9092, Cell Signaling Technology), rabbit monoclonal anti-Nanog (1:200, #9092, Cell Signaling Technology), mouse monoclonal anti-α-tubulin (1:200, T6199, Sigma-Aldrich), MF20c (1:50, DSHB), goat polyclonal anti-α-sarcoglycan (1:50, sc-16165, Santa Cruz Biotechnology), and mouse monoclonal anti-β-dystroglycan (1:500, NCL-b-DG, Leica Biosystems, Germany). Secondary antibody dilutions were goat anti-mouse AF488 (1:400, A11029, Thermo Fisher Scientific) and donkey anti-goat AF594 (1:400, A11058, Thermo Fisher Scientific). Finally, cells were mounted with ProLong Gold antifade reagent with DAPI (Invitrogen). Images were obtained with the Invitrogen EVOS FL auto 2 Cell Imaging System. Quantification of α-sarcoglycan expression in differentiated myotubes were done in ImageJ software v2.0 using line intensity plot profile of individual myotubes and normalized with respective DAPI intensity. Percentage of α-sarcoglycan intensity for each group were plotted and statistical analysis was done by Kruskal-Wallis multiple comparison test.

RNA Isolation and qPCR

Total RNA was extracted from hiPSCs with TRIzol (Thermo Fisher Scientific). The yield and quality of purified RNA samples were determined using NanoDrop 2000 Spectrophotometer (Thermo Scientific). 1 μg of total RNA samples were treated with DNase I (Invitrogen) for 15 mins and then heat inactivated with 2.5 mM EDTA at 65°C for 10 mins. DNase I treated total RNA was reverse transcribed with oligo dT primer using SuperScript III reverse transcriptase (Thermo Fisher Scientific). qPCR was performed in triplicate with Power SYBR Green PCR master mix (Applied Biosystems) in QuantStudio 3 Real-Time PCR System for MyoD1, MyoG, Nanog, and GAPDH. GAPDH was used as endogenous control. Relative expression levels were calculated by the cycle threshold method. Primer sequences used in qPCR are mentioned in Table S5.

Lentivirus Generation

For tamoxifen inducible MyoD expressing 3^rd generation lentivirus production, HEK293T cells were transfected at 80%–90% confluency with psPAX2, pMD2.G, and pCMVMyoD-(ERT)puro plasmids using Lipofectamine 3000 (Invitrogen). Lentiviral particles were harvested as supernatant after 48 h and 72 h of transfection. The psPAX2 and pMD2.G plasmids and the pCMVMyoD-(ERT)puro plasmid were a generous gift from Prof. Joseph A. Baur’s laboratory, UPenn, and Prof. M. Carrie Miceli’s laboratory, UCLA, respectively.

Directed Differentiation of hiPSC Lines

hiPSCs were differentiated into skeletal muscle cells by overexpression of MyoD, as described in Young et al.²² hiPSCs were plated as single cells on Matrigel in SMC4 (basal media: DMEM/F12 with 20% knock-out serum replacement (KOSR, Life Technologies), 1% Non-Essential Amino Acids Solution (NEAA, Life Technologies), 1% Glutamax (Life Technologies), 100 μM beta-mercaptoethanol, 10 ng/mL basic fibroblast growth factor (bFGF, Life Technologies), SMC4: basal media with daily addition of 5 μM ROCK inhibitor (Y27632, StemCell Technologies), 0.4 μM PD0325901 (Sigma-Aldrich), 1 μM CHIR99021 (Tocris Bioscience), and 2 μM SB431542 (Tocris Bioscience). When cells were 70%–80% confluent, they were infected with tamoxifen inducible MyoD-ERT lentivirus with 4 μg/mL protamine sulfate (Sigma-Aldrich) and spun inoculated at 1,250 rpm for 90 mins at 32°C. 48 h post-transduction cells were selected with 1 μg/mL puromycin in SMC4 for 3 days. Next day cells were split and plated on Matrigel in basal media without bFGF plus 5 μM ROCK inhibitor at approximately 1 × 10⁵ cells/cm². The cells were treated with 5 μM tamoxifen in DMEM with 15% FBS for 4 days for MyoD induction and then differentiated in low glucose DMEM with 5% horse serum and 1μM tamoxifen for 7 days with daily change of media.

Statistical Analysis

Data were analyzed using the GraphPad Prism v8 statistical software package. Values are presented as mean ± standard error of mean (SEM). Statistical analysis was performed using Mann-Whitney test or Kruskal-Wallis test with statistical significance set at p ≤0.05. For image quantification statistical analysis was performed using Kruskal-Wallis test with statistical significance set at p ≤0.05.

Author Contributions

T.S.K., M.K.M., and K.S. designed the study. K.S. and M.K.M. performed the experiments. K.S. and T.S.K. wrote the manuscript. T.S.K., A.D.P., M.J.S., E.L., M.K.M., and K.S. edited the paper. All authors discussed the study and approved submission.

Conflicts of Interest

T.S.K. is an inventor of several utrophin patents assigned to the University of Pennsylvania and subject to the institutional patent policies, which may include royalty distribution in the event of licensure. A.D.P. and M.J.S. are co-founders of MyoGene Bio.