REGENERATIVE MEDICINE

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1 REGENERATIVE MEDICINE Ex Vivo Gene Editing of the Dystrophin Gene in Muscle Stem Cells Mediated by Peptide Nucleic Acid Single Stranded Oligodeoxynucleotides Induces Stable Expression of Dystrophin in a Mouse Model for Duchenne Muscular Dystrophy FARNOOSH NIK-AHD, CARMEN BERTONI Key Words. Muscle stem cell Satellite cells Muscle progenitor cells Duchenne muscular dystrophy mdx 5cv Gene editing Gene repair Single stranded oligodeoxynucleotides Dystrophin Department of Neurology, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California, USA Correspondence: Carmen Bertoni, Ph.D., Department of Neurology, University of California Los Angeles, 710 Westwood Plaza, Los Angeles, California 90095, USA. cbertoni@ucla.edu Received October 14, 2013; accepted for publication January 26, 2014; first published online in STEM CELLS EXPRESS February 7, VC AlphaMed Press /2014/$30.00/ /stem.1668 ABSTRACT Duchenne muscular dystrophy (DMD) is a fatal disease caused by mutations in the dystrophin gene, which result in the complete absence of dystrophin protein throughout the body. Gene correction strategies hold promise to treating DMD. Our laboratory has previously demonstrated the ability of peptide nucleic acid single-stranded oligodeoxynucleotides (PNA-ssODNs) to permanently correct single-point mutations at the genomic level. In this study, we show that PNAssODNs can target and correct muscle satellite cells (SCs), a population of stem cells capable of self-renewing and differentiating into muscle fibers. When transplanted into skeletal muscles, SCs transfected with correcting PNA-ssODNs were able to engraft and to restore dystrophin expression. The number of dystrophin-positive fibers was shown to significantly increase over time. Expression was confirmed to be the result of the activation of a subpopulation of SCs that had undergone repair as demonstrated by immunofluorescence analyses of engrafted muscles using antibodies specific to full-length dystrophin transcripts and by genomic DNA analysis of dystrophin-positive fibers. Furthermore, the increase in dystrophin expression detected over time resulted in a significant improvement in muscle morphology. The ability of transplanted cells to return into quiescence and to activate upon demand was confirmed in all engrafted muscles following injury. These results demonstrate the feasibility of using gene editing strategies to target and correct SCs and further establish the therapeutic potential of this approach to permanently restore dystrophin expression into muscle of DMD patients. STEM CELLS 2014;32: INTRODUCTION Duchenne muscular dystrophy (DMD) is caused by mutations in the dystrophin gene, which result in a complete absence of dystrophin protein throughout the body [1 3]. Dystrophin deficiency greatly compromises the structural integrity of muscle tissue, and leads to rapid muscle necrosis, weakness, and ultimately, premature death between the second and third decades of life. The dystrophin protein contains four structural domains including the N- terminal, rod, cysteine-rich, and C-terminal domains, each playing key functional roles in the maintenance of membrane integrity. An understanding of the various defects in the dystrophin gene that cause DMD has enabled the development and testing of different therapeutic approaches [4, 5]. The majority of those approaches are aimed at restoring shorter, although still functional dystrophin protein. Preclinical and clinical studies have confirmed the beneficial effects that some of those strategies can achieve in animal models and DMD patients. For instance, the use of antisense oligonucleotides to redirect mrna splicing to restore protein expression has attained some success in restoring functional dystrophin protein in animal models for the disease as well as in clinical trials in DMD patients [6 9]. However, the level of expression is limited, as are the long-term beneficial effects. Strategies focused on restoring permanent levels of full-length dystrophin expression remain the best option to treat DMD [10 13]. Among those, readthrough of premature stop codons has shown some promise [14 16]. Gene correction mediated by single-stranded oligodeoxynucleotides (ssodns) in muscle cells has shown to induce single base-pair alterations STEM CELLS 2014;32: VC AlphaMed Press 2014

2 1818 Gene Editing in Muscle Stem Cells at the genomic level which are stably inherited through cell division [10 13]. Correction takes advantage of innate repair mechanisms present in the cells and capable of directing single base substitutions in the genomic DNA targeted for repair. We have recently demonstrated that ssodns composed of peptide nucleic acid bases (PNA-ssODNs) targeting the single base mutation in the mdx 5cv mouse are capable of achieving a higher frequency of gene correction than their unmodified counterparts rendering this technology a realistic possibility for the treatment of DMD [11, 13]. However, our results have also evidenced the presence of factors, other than correction frequencies, that contribute to the long-term stability of dystrophin expression into muscles following correction [13, 17]. In fact, the progressive loss of dystrophin expression detected after treatment in muscles that received ssodns compared to earlier time points clearly demonstrates that correction of mature myofibers alone is not sufficient to protect muscle from degeneration [13]. These results prompted us to test the ability of PNA-ssODNs to target and correct satellite cells (SCs) and to determine the ability of cells that had undergone correction to stably restore dystrophin expression following transplantation into muscle of mdx/nude mice [18, 19]. SCs are a class of stem cells found between the basal lamina and sarcolemma of muscle fibers [20]. They activate in response to injury or disease and are responsible for regenerating myofibers lost as a result of normal muscle turnover or in response to injury. Upon activation, SCs undergo an initial stage of cell division which results in the generation of two daughter cells. Of those, one will return to the quiescent stage while the other will continue to divide to give rise to progenies capable of fusing amongst each other to generate new myofibers or capable of fusing with preexisting fibers and repairing damaged ones. Here, we demonstrate that SCs isolated from a mouse model of DMD efficiently take up PNA-ssODNs and are amenable to gene repair. When transplanted into skeletal muscle of dystrophin-deficient mice, cells were able to restore low, although detectable, levels of dystrophin protein. Importantly, we demonstrate that the number of dystrophin-positive fibers significantly increases over time suggesting that correction of a portion of the transplanted cells is sufficient to actively regenerate muscle and to induce beneficial effects. These results clearly establish the importance of targeting SCs for the treatment of muscle disorders and pave the way for future studies aimed at determining the long-term therapeutic potential of cellmediated regenerative medicine for muscle disorders using gene editing strategies. MATERIALS AND METHODS Mice Mice of the mdx 5cv strain (B6Ros.Cg-Dmd mdx25cv /J) and control C57 strain (C57BL/6J) were used as donor mice to obtain mdx 5cv SCs and SCs isolated from wild-type mice, respectively. The mdx/nude strain was generated by backcrossing the mdx mouse with the nude mouse (CBy.Cg-Foxn1nu/J). All mice were purchased from The Jackson Laboratory (Bar Harbor, ME, All procedures were carried out in accordance with the guidelines of the Administrative Panel on Laboratory Animal Care of the University of California, Los Angeles. VC AlphaMed Press 2014 PNA-ssODN Synthesis The 18 bp control (PNA-CTL C ) and correcting (PNA-COR C ) oligonucleotides were designed to be complementary to the transcribed strand (Fig. 1A) and were synthesized by Panagene, Inc. (Panagene, North Korea, com) [13]. All oligonucleotides were HPLC purified and exhibited a single peak of the expected molecular weight as determined by MALDI TOF mass spectroscopy analysis. SC Isolation and Transfection SCs were isolated from hind limb muscles of mdx 5cv mice (6 8 weeks of age) as described previously [10, 21, 22]. Briefly, muscles were incubated in Dulbecco s modified Eagle s medium (DMEM) with 0.2% (wt/vol) collagenase II (Life Technologies, Carlsbad, CA, SCs were released from bulk fibers through an additional digestion in 20 ml of Hams F-10, 10% horse serum (HS), 0.5 U/ml dispase (Life Technologies), and 38 U/ml collagenase type II (U.S. Biological, Cells were sedimented by centrifugation and the supernatant containing the SCs was filtered and centrifuged. The pellet was then resuspended in growth medium consisting of Ham s F-10 nutrient mixture (Mediatech, Herndon, VA, supplemented with 20% fetal bovine serum (FBS), penicillin, and streptomycin and plated in six-well plates ( cells/well) coated with extracellular matrix (Sigma, St. Louis, MO, 1:500). At this stage, the preparation contained primarily SCs as determined by flow cytometry and immunostaining analyses (Fig. 1 and Supporting Information Fig. S1). Cells were transfected 2 hours later with PNAssODNs (150 pmol/ml) as previously described [13]. SCs isolated from age-matched C57 mice were used as a positive control, and received a sham transfection to control for the procedure. Cell differentiation was induced by maintaining the cells in low serum medium (differentiation medium) consisting of DMEM supplemented with 2% HS, penicillin, and streptomycin. Single fiber cultures were prepared as described previously [23]. Tibialis-anterior (TA) muscles of transplanted mice were digested in collagenase II and single fibers were cultured in proliferation medium containing 20% FBS (Mediatech) and bfgf for 24 hours prior to fixation and immunostaining analysis. Flow Cytometry Analyses were performed using a Beckton Dickinson FACScalibur flow cytometry (Becton Dickinson, Franklin Lakes, NJ). Uptake of oligonucleotides was measured in cells trypsinized and harvested 24 hours after transfection. Analyses of intracellular epitopes were performed in cells fixed with 4% paraformaldehyde and permeabilized with staining buffer containing 0.1% Triton X-100. Cells were stained with an antirat CD34 antibody conjugated to FITC (Beckton-Dickinson, San Jose, CA, 1:100). Data were acquired at events per sample. Cells were distinguished from noncellular debris using forward and side scatter gating. Cell Grafting and Muscle Harvesting Hind limbs of host mice (6 8 weeks of age) were irradiated with 18 Gy and mice were allowed to recover for 4 days [24 STEM CELLS

3 Nik-Ahd, Bertoni 1819 Figure 1. Isolation of SCs and transfection. (A): The A-to-T mutation in exon 10 of the mdx 5cv dystrophin gene responsible for the absence of dystrophin is underlined. The targeting (PNA-COR C ) and nontargeting (PNA-CTL C ) ssodns are perfectly homologous to the region of the dystrophin gene targeted for repair with the exception of a single base-pair mismatch between the targeting oligonucleotide and the mdx 5cv mutation. (B): Density plot of myofiber associated SCs isolated from lower limb muscles of mice. Cells were gated based on cell granularity (side scatter) and cell size (forward scatter) for all subsequent analyses. (C): Cells were analyzed 2 hours after isolation for the expression of CD34, a marker of SCs. (D): Cells isolated from mdx 5cv and wild-type muscles were analyzed for the expression of Pax7, MyoD, desmin, and myogenin. A significant difference in the percentage of cells expressing MyoD, desmin, and myogenin was detected in all samples analyzed and isolated from muscle of mdx 5cv mice compared to samples obtained from age-matched wild-type controls. Results were consistent among triplicate experiments. Values are presented as mean 6 SD (*, p <.005). (E): Fluorescently labeled PNA-ssODNs were transfected into mdx 5cv SCs 2 hours after isolation and were analyzed for CY3-uptake 22 hours after transfection. Histogram-plots of CY3-positive cells 1 day after ssodn transfection demonstrated that nearly 100% of the cells had takenup the oligonucleotide. Abbreviations: PNA, peptide nucleic acid; SC, satellite cell; ssodn, single-stranded oligodeoxynucleotide. 26]. To promote regeneration, TA muscles of recipient mice were injured 4 days prior to engraftment using a single intramuscular injection (50 ml) of cardiotoxin (Calbiochem, Gibbstown, NJ, resuspended at a concentration of 100 ng/ml [27, 28]. Three days after cardiotoxin injection, cells were engrafted into hind limbs of host mice. All transplantation procedures were performed using cells maintained in growth media for 12 hours following explant. Plates were washed three times with phosphate buffered saline (PBS) to eliminate debris and nonadherent cells. Cells were then trypsinized, centrifuged, and resuspended in a final volume of 50 ml of injectable grade saline solution. VC AlphaMed Press 2014

4 1820 Gene Editing in Muscle Stem Cells Following the initial dose optimization study (Fig. 3C), all subsequent transplantation procedures were performed using a single batch of cells prepared from mdx 5cv or wild-type mice and at a dose of 5,000 cells per engrafted muscle. Some mice received a second injection (50 ml) of cardiotoxin (100 ng/ml), 3 weeks after grafting, in ipsilateral TA muscles. TA muscles were isolated, embedded in Tissue-Tek O.C.T compound (Sakura Finetek U.S., Inc., Torrance, CA, com) and muscles were transversely cut along the longitudinal axis into sections of 10 mm thickness at intervals of 300 mm and mounted on slides. Immunofluorescence and Western Blot Analyses Immunostaining analyses of cells in culture were performed using mouse antibodies to Pax7 (Developmental Studies Hybridoma Bank, Iowa City, IA, 1:100), MyoD (BD Pharmingen, San Jose, CA; 1:250), desmin (Sigma; 1:500), and myogenin (BD Pharmingen, San Jose, CA, 1:250), incubated overnight, and counterstained with an Alexa 488-conjugated goat-antimouse immunoglobulin (Ig) (Life Technologies). Single fibers isolated from TAs of mdx/nude mice engrafted with SCs were permeabilized with 0.5% Triton X-100 (Sigma), blocked with 20% goat serum, and incubated overnight with a mouse-anti-pax7 antibody. Myofibers were counterstained with an Alexa 488-conjugated goat-anti-mouse Ig (Life Technologies) secondary antibody. Dystrophin immunostaining of cultured cells was performed as previously described [10, 11, 13] using a monoclonal antibody (Mandys- 8, Sigma; 1:200) followed by an Alexa 546-coupled goat-antimouse (H1L) (Life Technologies; 1:250). For immunohistology, sections were incubated using a polyclonal antibody against dystrophin (Thermo Scientific Neomarkers, Fremont, CA, 1:100) and detected with the Alexa 546-coupled goat-anti-rabbit secondary antibody (Life Technologies; 1:500) [29]. Hoechst staining (1:15,000) was used to visualize the nuclei within myofibers. Consecutive sections isolated from injected muscles were immunoassayed using Mandys-1011 (1:50) or Mandys-18 (1:100) to confirm the expression of full-length dystrophin [11, 13, 30]. Specific antibody binding was detected with the Alexa 546-coupled goat-anti-mouse secondary antibody (1:1,000). Reduction of nonspecific binding of the secondary antibody was achieved using a papain digested whole goat-anti-mouse antibody at a concentration of 25 mg/ ml as previously described [13, 30, 31]. For the detection of quiescent and activated SCs, cryosections of TA muscles (10 mm) were incubated with a rabbit polyclonal antibody against MyoD (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, 1:200) and mouse monoclonal antibody against Pax7 (Hybridoma Bank; 1:100) labeled with a Zenon labeling kit (Life Technologies) according to manufacturers instructions. Rabbit polyclonal antibodies were detected with the Alexa 546-coupled goat-anti-rabbit secondary antibody as described above. Western blot analysis was performed using an antibody directed toward the rod domain (Mandys-8; 1:400) of the dystrophin protein [10, 11, 32]. a-actin was used as a sample loading control and was detected using a rabbit-anti-actin antibody (Sigma). VC AlphaMed Press 2014 DNA and RNA Analyses Gene correction was assessed in all cultures used for the transplantation procedures using cells maintained in culture for up to 2 weeks following transfection of the control and correcting PNA-ssODNs. Analyses at the genomic level were performed using DNA (200 ng) digested with HphI and subjected to amplification using the Forw-ex 10 primer and Revint 10 primers or the Forw-ex 22 primer and Rev-int 23 primers (Fig. 2C and Supporting Information Table S1) [11, 13]. Analysis of mrna transcripts was performed using previously described protocols [11, 13]. Polymerase chain reactions (PCRs) on reverse transcribed cdna (PCR) reactions were carried out using the forward primer (Forw-ex 9 ) and the reverse primer (Rev-ex 10in ) (Fig. 2D and Supporting Information Table S1). The ratios of gene correction obtained by targeting PNAssODNs to targeting ssodns were calculated using a standard DDCt method (2 2DDCt ) [33]. All data were normalized to GAPDH which was used as control [17, 34]. All reactions were performed on a MyiQ single-color detection system (Bio-Rad Laboratories, Hercules, CA, as described [13]. Amplicons were separated on 1.5% agarose gels and PCR products were purified using the Qiagen gel extraction kit (Qiagen). DNA sequencing was carried out using an Applied Biosystems ABI377 automated sequencer. Quantitative Measurements of Muscle Fibers The number of central nuclei in muscles was determined in sections immunostained for dystrophin and counterstained with Dapi (Life Technologies). A minimum of 500 fibers per muscle were analyzed for each treatment group. Fibers were considered to have centrally-located nuclei if there was at least one nucleus within the central portion of the dystrophin-positive fiber [29]. The relative amount of dystrophin expression restored within individual myofibers was measured in muscle sections immunoassayed for dystrophin using an Applied Imaging Ariol SL-50 [35]. For each region, the minimum intensity value recorded (representative of the cytoplasm or background intensity) was subtracted from the maximum intensity value (corresponding to the sarcolemma) to correct each measurement for background intensity. Values are reported as percentage of the control. Statistical Analyses Data are presented as means and standard deviations (SDs). Comparisons between groups were done using Student s t test assuming two-tailed distribution and equal variances. RESULTS Oligonucleotide Design and Transfection of SCs SCs were isolated from purified single fibers obtained by enzymatic digestion of mdx 5cv muscles followed by gentle trituration and dissociation [36, 37]. Flow cytometry was used to assess the purity of the preparation and demonstrated that the majority of the cells expressed CD34, a marker of SCs (Fig. 1B, 1C). Immunostaining analyses confirmed the purity of the STEM CELLS

5 Nik-Ahd, Bertoni 1821 Figure 2. Analysis of gene correction in vitro. SCs transfected with targeting or control PNA-ssODNs were analyzed for the expression of dystrophin 2 weeks after isolation. (A): Dystrophin was clearly detected in cells treated with the PNA-COR C and was absent in cells transfected with the control ssodn (scale bar 5 50 mm). (B): Western blot analysis revealed the expression of full-length dystrophin only in cells transfected with the targeting oligonucleotide. Proteins isolated from wild-type myotubes were mixed with total protein isolated from mdx 5cv myotubes at a ratio of 1:20 and were used as a control. Equal loading was confirmed using an antibody specific to a- actinin. (C): Expression of full-length dystrophin was assessed by RT-polymerase chain reaction (PCR) in total mrna isolated from cells transfected with targeting or control oligonucleotides using a reverse primer positioned in exon 10 and complementary to the region of the dystrophin mrna spliced out in the mature mrna and a forward primer homologous to exon 9. No amplification was detected in untreated cells or in cells treated with the control PNA-ssODNs. A band of the predicted molecular weight was clearly detected only in cells transfected with PNA-COR C. All amplicons were normalized to GAPDH. (D): Gene correction was analyzed by real-time PCR using DNA isolated from cells and subjected to HphI digestion. Amplicons were resolved by agarose gel electrophoresis and revealed the presence of a specific PCR product only in cells treated with PNA-COR C but not in cells treated with PNA-CTL C. GAPDH was used as an internal standard control. Abbreviations: PNA, peptide nucleic acid; SC, satellite cell. preparation and also revealed the presence of distinct populations of myogenic cells in preparations obtained from muscles of mdx 5cv mice compared to those obtained from wild-type mice (Fig. 1D and Supporting Information Fig. S1). The significantly higher percentage of cells expressing MyoD, desmin, and myogenin detected in muscles isolated from mdx 5cv mice are likely to be due to the degenerative process that characterizes muscles lacking dystrophin and that results in the activation of SCs that are responsible for replacing myofibers that have degenerated as also observed by others [38]. Flow cytometry analysis clearly demonstrated that fluorescently labeled PNA-ssODNs are efficiently delivered into cells VC AlphaMed Press 2014

6 1822 Gene Editing in Muscle Stem Cells maintained in culture for 2 hours following isolation and subsequently transfected for 10 hours (Fig. 1E and Supporting Information Fig. S2). Time course analyses demonstrated that approximately 95% of the transfected cells had taken-up the PNA-ssODNs when analyzed 24 hours following transfection. Fluorescence remained high for at least 48 hours, but rapidly declined between 72 and 96 hours following oligonucleotide delivery (Supporting Information Fig. S2B). Gene Correction Mediated by PNA-ssODNs In Vitro The frequencies of gene repair achieved in SC cultures were assessed 2 weeks following isolation and transfection. Immunostaining analyses revealed the expression of dystrophin in some of the cells propagated in vitro and then induced to differentiate for 48 hours (Fig. 2A). Results were confirmed by Western blot analyses (Fig. 2B). Dystrophin was absent in untransfected cells or cells treated with PNA-CTL C. Cells transfected with PNA-COR C showed a clear band at the expected molecular weight although expression was much lower than that obtained from SCs isolated from wild-type muscles and maintained in culture for the same period of time (Fig. 2B). Quantitative analysis of protein expression obtained from multiple independent experiments demonstrated that the level of full-length dystrophin protein ranged between 1% and 2% of that of wild-type cells (mean 1.7% %). Real-time PCR was used to validate the results obtained by Western blot analysis (Fig. 2C). A band of the exact molecular weight as that obtained in cells isolated from wild-type mice was clearly detected only in mdx 5cv cells that received PNA- COR C, but not in cells treated with the control PNA-ssODN, further confirming the specificity of the correction process mediated by targeting oligonucleotides (Fig. 2C). The level of full-length dystrophin expression detected was similar to that obtained by Western blot analysis (mean 1.5% %). To further confirm that the results obtained were due to correction of the dystrophin gene, genomic DNA was isolated from cells treated with the targeting or control PNA-ssODNs and subjected to restriction DNA digestion to abrogate all copies of the dystrophin mdx 5cv gene refractory to correction (Fig. 2D). Amplification of a specific product was detected in mdx 5cv cells transfected with the targeting oligonucleotide, but not in cells treated with the control PNA-ssODN. Direct sequencing of the amplicon confirmed correction at the genomic DNA level (not shown). The frequencies of gene repair ranged between 1.5% and 2.1% as determined by quantitative PCR thus confirming the results obtained at the mrna and protein levels. Altogether these results demonstrate that PNA-COR C can target and induce the desired single base-pair alteration at the DNA level and that correction results in the expression of full-length dystrophin. Transplantation of SCs in mdx/nude Mice VC AlphaMed Press 2014 A dose response study was used to assess the ability of SCs that had undergone gene repair to restore dystrophin expression in dystrophin-deficient mice. Experiments were conducted in the mdx/nude strain, a known mouse model for DMD. This strain has been used extensively in the field and has shown to provide efficient engraftment following transplantation. Furthermore, the impaired immune system in immunocompromised mdx/nude mice has been associated with reduced fibrosis rendering comparative analyses between individual experiments more reliable especially in older mice [18, 19]. To determine the minimum number of SCs needed to be transplanted in order to detect an effect, we isolated SCs from mdx 5cv mice, allowed them to seed for 2 hours and then transfected them with the targeting PNA-ssODN as described above (Fig. 3A). As positive control, we used SCs obtained from wild-type mice that had undergone the same isolation procedure, but that were treated with transfection reagent alone [27, 28]. Dystrophin-positive fibers were clearly detected in all muscles that received SCs (Fig. 3B, 3C). No statistically significant differences were detected in the number of dystrophin-positive fibers in muscles that received mdx 5cv SCs treated with PNA-COR C at any of the dosages analyzed (Fig. 3C). These results suggested that, under our experimental procedures, high dosages of SCs are required to achieve limited, although detectable, dystrophin expression in muscle. Therefore, all subsequent analyses were performed using a dose of 5,000 cells per transplanted TA. Retention of Stem Cell Properties Following In Vitro Transfection with PNA-ssODNs A hallmark characteristic of any stem cell population is the ability to emerge from a quiescent state in response to the needs of its environment and to divide to produce daughter cells which are ultimately responsible for repairing injured or diseased myofibers. To better characterize the effects of transplanting SCs which have been transfected with oligonucleotides, we injured TA muscles of engrafted mice and analyzed them for the expression of dystrophin 2 weeks after injury (Fig. 4A) [26, 39, 40]. An increase in the number of dystrophin-positive fibers was clearly detected in muscles that had undergone injury following transplantation compared to uninjured muscles (Fig. 4B). No significant differences in the number of dystrophin-positive fibers were observed in muscles of irradiated mice that were subjected to cardiotoxin injection 3 weeks following irradiation and analyzed 2 weeks later (Supporting Information Fig. S3). Immunostaining analyses were used to assess for the presence of activated and differentiated SCs. Sections isolated from engrafted muscles were immunoassayed for the expression of Pax7 and MyoD, markers of quiescent and activated SCs, respectively. Pax7-positive cells were clearly detected in muscles of control mice or muscles of mdx/nude mice engrafted with SCs, but not in muscles of irradiated mice that did not receive SCs and that underwent cardiotoxin injury (Fig. 4C). Expression was confined to few isolated cells located adjacent to the muscle membrane. The majority of those cells coexpressed Pax7 and MyoD, suggesting that engrafted cells had retained the ability to divide and to give rise to muscle progenitor cells (Fig. 4D and Supporting Information Fig. S4). To further clarify the effects of SC activation and repair in muscles, we analyzed consecutive sections of isolated TAs and followed the distribution of dystrophin-positive fibers along the length of the engrafted muscles (Fig. 5). On average, the longitudinal distribution of dystrophin in muscles that had undergone cardiotoxin injury following transplantation was greater than that detected in resting muscles and was confirmed in both, muscles that received mdx 5cv SCs transfected with the targeting PNA-ssODN as well as in muscles engrafted with SCs isolated from wild-type mice. Moreover, the number STEM CELLS

7 Nik-Ahd, Bertoni 1823 Figure 3. Dystrophin expression following transplantation of SCs. (A): Experimental strategy used to isolate, transfect, and engraft SCs into recipient muscles. (B): Clusters of dystrophin-positive fibers were consistently observed in muscles injected with mdx 5cv SCs transfected with PNA-single-stranded oligodeoxynucleotides (ssodns) or SCs isolated from wild-type muscle at all the dosages analyzed. Muscles isolated from mdx/nude mice only showed scattered revertant fibers immunoreactive to dystrophin (scale bar mm). (C): Average number of dystrophin-positive fibers per cross-sectional area containing the highest number of positives. A significant increase in the number of dystrophin-positive fibers was only detected in muscles of mdx/nude mice that received the highest dose of SCs obtained from wild-type muscle compared to all other dosages and treatment types. Muscles that received mdx 5cv SCs transfected with PNA-ssODNs did not show a significant difference in the number of dystrophin-positive fibers at any of the dosages tested (*, p.02; **, p <.005, n 5 4 muscles per treatment group). Values are mean 6 SD. Abbreviations: PNA, peptide nucleic acid; SC, satellite cell. of fibers comprising each individual cluster expressing dystrophin was one- to two-fold higher than that observed in muscles that received SCs but that had not undergone injury following transplantation (Fig. 5B, 5C). Altogether, these results provide strong evidence that at least a few of the engrafted SCs had maintained their ability to return to quiescence and that, when prompted, they were able to exit the quiescent state and actively contribute to muscle regeneration. Persistence of Dystrophin Expression Over Prolonged Periods of Time The long-term therapeutic application of gene correction in SCs was assessed 24 weeks after transplantation in mice that received mdx 5cv SCs transfected with PNA-COR C and compared to that achieved after engraftment of SCs isolated from wildtype muscle. Large clusters of dystrophin-positive fibers were detected in all muscles that received SCs, but not in TAs of untreated mice (Fig. 6A). Interestingly, the number of dystrophin-positive fibers detected in muscles that received mdx 5cv SCs treated with the targeting PNA-ssODN was threefold higher than that observed 5 weeks after transplantation suggesting that SCs that had undergone gene repair were able to actively contribute to muscle regeneration (Fig. 6B). To further confirm that dystrophin-positive fibers detected in transplanted muscles expressed full-length dystrophin, we immunoassayed consecutive sections isolated from engrafted muscles with an antibody specific to a region of the dystrophin protein that is spliced out as a result of the mdx 5cv mutation (Mandys-1011) or with an antibody recognizing the region of the dystrophin protein close to the mdx/nude mutation responsible for the absence of dystrophin in this strain (Mandys-18) [11, 30, 41, 42]. TAs isolated from age-matched mdx/nude mice showed small clusters of dystrophin-positive VC AlphaMed Press 2014

8 Figure 4. Activation of SCs in response to injury. (A): Ipsilateral and contralateral tibialis-anterior (TA) muscles were transplanted with mdx 5cv SCs transfected with PNA-COR C or SCs isolated from wild-type muscle at a dosage of 5,000 cells per engrafted TA and a single injection of cardiotoxin was administered on the ipsilateral TA, 3 weeks after engraftment. Mice were allowed to recover for 2 weeks prior to analyses. (B): The number of dystrophin-positive fibers remained significantly higher in muscles that received SCs obtained from C57/BL10 mice compared to muscles engrafted with mdx 5cv SCs transfected with the targeting PNA-single-stranded oligodeoxynucleotide (ssodn) or sham-injected muscles. No significant differences were detected in TAs that received mdx 5cv SCs treated with PNA-COR C compared to control muscles. Values are presented as mean 6 SD (*, p 5.04; **, p.002, n 5 3 muscles per treatment group). (C): Sections were immunoassayed with a FITC-labeled Pax7 antibody and counterstained with an antibody specific to MyoD (labeled in red) and Dapi (labeled in blue). A subset of the cells positive for Pax7 were also positive for MyoD (yellow arrow). MyoD-positive, Pax7-negative cells (white arrow) were also evident in those muscles (scale bar 5 50 mm). (D): Immunostaining analysis of muscles transplanted with SCs and injured with cardiotoxin 3 weeks after transplantation. Immunostaining was performed using a polyclonal antibody to dystrophin (labeled in red) that was used to localize the area of the muscle engrafted with mdx 5cv SC treated with the targeting PNA-ssODN that had undergone gene repair or engrafted with SCs isolated from wild-type mice and a polyclonal antibody to MyoD (also labeled in red) used to visualize muscle progenitor cells resulting from the activation and division of SCs. Sections were counterstained with a FITC-labeled Pax7 monoclonal antibody (labeled in green) and Dapi (labeled in blue). Pax7-positive, MyoD-negative cells were evident in close proximity of dystrophin-positive fibers. Results were consistent in at least three different muscles per treatment group analyzed (scale bar 5 50 mm). Abbreviations: PNA, peptide nucleic acid; SC, satellite cell.

9 Nik-Ahd, Bertoni 1825 fibers immunoreactive to Mandys-1011 and limited or no expression was observed following analysis using an antibody specific to exon 32 of the dystrophin protein (Supporting Information Fig. S6). Virtually all the fibers that were immunoreactive to the region of dystrophin encoded by exons 10 and 11 were also recognized by the antibody raised against the more distal regions of the dystrophin protein (Supporting Information Fig. S6). Results were confirmed at the molecular level (Supporting Information Fig. S7). Sections obtained from muscles engrafted with SCs were immunostained for dystrophin and dystrophinpositive fibers were mechanically excised. Total genomic DNA was isolated from purified fibers using standard methods and was subjected to HphI digestion to eliminate all copies of dystrophin DNA that could have arisen, in those fibers, from the contribution of noncorrected mdx 5cv SCs [11, 13]. Amplification was carried out using a set of primers encompassing exon/ intron 10 and a separate set of primers positioned across intron 22 and exon 23 of the dystrophin gene. Amplification of a product was obtained in all muscles isolated from untreated mdx/nude mice or muscles engrafted with SCs, but not in TAs isolated from mdx 5cv mice, confirming the specificity of the amplification products. Direct sequencing of the PCR amplicons confirmed the presence of corrected sequences containing the desired T-to-A transversion in exon 10 of the dystrophin gene and lacking the G-to-T mutation characteristic of the mdx/nude mouse (Supporting Information, Fig. S7). To ensure that the differences in dystrophin-positive fibers detected in muscles that received mdx 5cv SCs transfected with the targeting PNA-ssODNs compared to those observed in muscles that received wild-type SCs were not the result of differences in engraftment or survival of cells that could have occurred over time, we determined the number of quiescent and activated SCs in muscles isolated 24 weeks after SC transplantation (Fig. 6C 6E). No statically significant differences were detected in the number of Pax7- or MyoD-positive cells among muscles engrafted with SCs suggesting that the results obtained in muscles that received mdx 5cv SCs treated with the PNAssODN were not due to increases in the ability of cells that had undergone correction to home into muscle following transplantation and over prolonged periods of time (Fig. 6C, 6D). To gather further evidence that the increase in the number and distribution of dystrophin-positive fibers was the result of the contribution of SCs that had undergone gene repair, single fibers were isolated from engrafted muscles and analyzed for the expression of Pax7. Virtually no positives were detected in irradiated muscles of mdx/nude mice confirming the efficacy of the irradiation procedure to ablate SCs. In net contrast, Pax7- positive donor-derived SCs were evident in myofibers maintained in culture for 48 hours after explant and isolated from muscles engrafted with mdx 5cv SCs treated with PNA-COR C (Fig. 6F). Similar results were obtained from muscles isolated from control mice that received wild-type SCs (not shown). Figure 5. Distribution of dystrophin expression in transplanted muscles. Tibialis-anterior muscles were cut for at least two-thirds of the muscle length at intervals of 300 mm and examined for dystrophin expression. Clusters of dystrophin expression were traced along the length of the muscle. The majority of the dystrophin-positive fibers detected in muscles of non-irradiated mice injected with saline alone were scattered in isolated fibers that could not be traced for more than one section (A). The percentage of dystrophin-positive fibers showing greater than 900 mm in length was prominent in muscles that received mdx 5cv satellite cells (SCs) transfected with the targeting peptide nucleic acid single-stranded oligodeoxynucleotide (B) or SCs isolated from wild-type muscle (C). An increase in the distribution of dystrophin expression was clearly detected in all muscles in response to injury, presumably as the result of the activation of SCs which mediated the repair process. Values are expressed as mean 6 SD (*, p 5.03; **, p 5.004; n muscles per treatment group). VC AlphaMed Press 2014

10 Figure 6. Long-term persistence of dystrophin expression after transplantation. Muscles of mdx/nude mice were engrafted with 5,000 cells and muscles were analyzed 24 weeks following transplantation. (A): Clusters of dystrophin-positive fibers were evident in muscles engrafted with mdx 5cv SCs transfected with the targeting PNA-single-stranded oligodeoxynucleotide (ssodn) as well as in muscles that received SCs isolated from wild-type muscle (scale bar 5 50 mm). (B): Average number of dystrophin-positive fibers per cross-sectional area containing the highest number of positives. Quantitative analysis revealed a significant increase in the number of dystrophinpositive fibers in muscles injected with mdx 5cv SCs transfected with PNA-ssODNs compared to untreated mdx/nude muscles. Muscles injected with SCs isolated from control mice showed a two-fold increase in the number of dystrophin-positive fibers compared to those that received mdx 5cv SCs transfected with PNA-ssODNs (*, p.03; n 5 4 muscles per treatment group). (C): Average number of Pax7- positive cells detected in cross-sections randomly chosen among experimental groups (*, p 5.02; **, p 5.004; n 5 3 randomly chosen sections per treatment group). (D): No statistically significant differences were detected in the percentage of cells positive for both, Pax7 and MyoD of sections of muscles isolated from tibialis-anteriors engrafted with mdx 5cv SCs treated with the targeting PNA-ssODN compared to muscles that received wild-type SCs (*, p.05; n 5 3 randomly chosen sections per treatment group). (E): The presence of donor-derived SCs was assessed in engrafted muscles following immunostaining with Pax7 (labeled in green) and MyoD (labeled in red) and counterstained with Dapi (labeled in blue). White arrow indicates Pax7-positive cells, while yellow arrows indicate MyoDpositives (scale bar 5 10 mm). (F): Single fibers from mdx/nude mice engrafted with mdx 5cv SCs treated with PNA-COR C stained with anti-pax7 (labeled in red) and counterstained with Dapi (labeled in blue). White arrow indicates Pax7-positive cells. Results were confirmed in triplicate experiments (scale bar 5 50 mm). Abbreviations: PNA, peptide nucleic acid; SC, satellite cell.

11 Nik-Ahd, Bertoni 1827 Figure 7. Morphology of muscles 24 weeks following SC transplantation. (A): Immunohistochemical analyses of tibialis-anterior muscles engrafted with mdx 5cv SCs transfected with PNA-COR C or wild-type SCs and assayed for dystrophin (labeled in red) and Dapi (labeled in blue) (scale bar mm). (B): The percentage of dystrophin-positive fibers containing central nuclei was significantly lower in muscles that received mdx 5cv SCs transfected with PNA-COR C or SCs isolated from wild-type mice and that were analyzed 24 weeks after transplantation compared to that detected 5 weeks after engraftment (*, p.005; n 5 4 muscles per treatment group). (C): Average length of dystrophin-positive clusters in muscles that received mdx 5cv SCs transfected with the targeting PNA-ssODNs or SCs isolated from wildtype muscle (*, p 5.04; n 5 4 muscles per treatment group). All values are expressed as mean 6 SD. Abbreviations: PNA, peptide nucleic acid; SC, satellite cell. Morphological Analyses of Engrafted Muscles Following Transplantation of SCs To better characterize the functional activity of SCs that had undergone repair ex vivo and that were transplanted into recipient muscles, we analyzed the localization of myonuclei in dystrophin-positive fibers and used it as an index of functional recovery [43]. As expected, dystrophin-positive fibers isolated from muscles that received mdx 5cv SCs transfected with the targeting PNA-ssODN or SCs isolated from wild-type muscle that were analyzed 5 weeks after transplantation all contained predominantly central nuclei in dystrophin-positive fibers demonstrating that those fibers were newly regenerated and/or repaired (Fig. 7A, 7B). A significant decrease in the percentage of dystrophin-positive fibers containing central nuclei was evident 24 weeks after engraftment (Fig. 7B). No statistically significant differences were detected in the percentage of dystrophin-positive fibers containing central nuclei in muscles that received mdx 5cv SCs transfected with PNA-COR C compared to muscles that received SCs isolated from wild-type muscle. To gather further insights into the stability of dystrophin expression obtained over time and to determine whether the improvement in muscle morphology observed in muscles that received mdx 5cv SCs transfected with PNA-COR C could be correlated to the level of dystrophin expressed within individual fibers as the result of the contribution of transplanted cells, we analyzed the distribution of dystrophin expression along the length of the muscle (Fig. 7C). In muscles that received wildtype SCs, the majority of the dystrophin-positive fibers distributed for greater than two-thirds of the length of the muscle suggesting that, over time, engrafted SCs were able to actively contribute to muscle regeneration. A shift in the distribution of positive fibers expressing dystrophin was also observed in muscles that received mdx 5cv SCs transfected with PNA-COR C analyzed 24 weeks after transplantation compared to that detected 5 weeks after engraftment. Results were correlated to the level of dystrophin expression achieved within individual VC AlphaMed Press 2014

12 1828 Gene Editing in Muscle Stem Cells fibers (Supporting Information Fig. S8). An increase in the percentage of dystrophin expression was clearly detected in muscles that received SCs and that were analyzed 24 weeks after transplantation compared to that detected 5 weeks after transplantation. Importantly, at the later time point analyzed, the majority of the fibers that expressed dystrophin showed levels of expression equal to or greater than 50% of the intensity of dystrophin detected in wild-type mice. These results greatly support the hypothesis that the increase in dystrophin expression detected in muscles analyzed 24 weeks after engraftment was the result of the contribution of SCs and provide clues on the levels of dystrophin expression that can be achieved into muscle using regenerative approaches aimed at targeting SCs. DISCUSSION We have demonstrated the feasibility of using PNA-ssODNs to direct single-point mutations at the genomic level and to correct SCs ex vivo. Gene correction frequencies were lower than those detected in myoblasts and previously reported [13] suggesting the presence of intrinsic differences between those two cell populations. Differences in fluorescence intensity and persistence of fluorescently labeled PNA-ssODNs detected in SCs transfected in vitro compared to that previously observed in myoblasts treated under the same experimental procedures may in part be responsible for the differences in gene correction frequencies achieved in this study (Supporting Information Fig. S2). Similarly, differences in chromatin structure and chromatin folding typical of nondividing cells such as quiescent SCs may have restricted the accessibility of the ssodn to the region targeted and could in part be responsible for the low levels of gene repair observed. Nonetheless, correction was clearly detected in cells maintained in culture for up to 2 weeks following SC explant and was stably inherited through cell division. The significant increase in the number of cells that expressed MyoD, desmin, and myogenin obtained from mdx 5cv muscles compared to those detected in cultures of wild-type muscles demonstrated the presence of distinct populations of cells in our preparations including differentiated and committed muscle progenitors (Fig. 1D). This increase is likely to be the result of the degenerative process that characterizes dystrophic muscles and that leads to continuous rounds of SC activation necessary to form new myofibers. Accordingly, correction mediated by PNA-COR C may have occurred not only into quiescent SCs but also into activated SCs and muscle progenitor cells. The possibility that the expression of dystrophin detected following engraftment of cells isolated from mdx 5cv mice transfected with PNA-COR C may be the result of a small fraction of the muscle progenitor cells present in isolated muscles that had undergone repair rather than SCs is highly unlikely. First, extensive data in the literature have demonstrated that the number of cells required to achieve an effect are at least 1 order of magnitude higher than those used in this study [29, 40, 44]. Second, it has been clearly demonstrated that the majority of differentiated muscle progenitor cells die shortly after transplantation [45 47]. Furthermore, the increase in the number of dystrophin-positive fibers detected 24 weeks following transplantation in muscle that VC AlphaMed Press 2014 received mdx 5cv SCs treated with the targeting PNA-ssODN suggests that a large number of the corrected cells were able to proliferate. Although muscle progenitors capable of selfrenewing have been described they represent only a small minority of the cell population [48]. Thus, it is safe to conclude that the results obtained are likely to be due to a fraction of the SCs that had undergone repair, homed into muscles, and actively contributed to myofiber regeneration. Moreover, the results demonstrate that explant of SCs ex vivo and transfection in vitro does not compromise the ability of cells to engraft into muscles and to activate in response to injury or during homeostasis establishing the importance of targeting SCs for the treatment of DMD. Whether activated SCs that were present in the preparation obtained from mdx 5cv muscles are amenable to gene repair and were able to return into quiescence following transplantation into mdx/ nude mice remains to be established and is the focus of active studies in the laboratory. Most of the research aimed at determining the efficacy of SCs in restoring dystrophin expression for the treatment of DMD following engraftment has implemented the use of reporter systems to identify donor cells into transplanted muscles [49 52]. Although these systems have proven to be valuable tools, expression of proteins like b-galactosidase (bgal) or green fluorescent protein as experimental controls has been associated with a strong proinflammatory response toward the reporter protein and increased muscle damage rendering the assessment of the long-term therapeutic efficacy of SC-mediated regenerative approaches to DMD difficult to perform [53]. Our experimental settings overcome these limitations by ensuring that the expression of dystrophin detected following engraftment of mdx 5cv SCs treated with the targeting PNA-ssODN is the result of correction that has occurred at the genomic level and not the result of expansion of revertant fibers that may have occurred over time. First, muscles subjected to transplantation were irradiated to ablate all endogenous SCs. Although it has been previously shown that a fraction of SCs survive the procedure, these cells are extremely rare in mdx mice [54, 55]. The fact that the number of dystrophin-positive fibers in TA muscles of control mdx/ nude mice that had undergone irradiation remained practically unchanged over the course of the analyses compared to non-irradiated mdx/nude control muscles clearly demonstrates that the majority of the SCs are radiation sensitive and did not survive the procedure (Supporting Information Figs. S3, S5). Second, expression of full-length dystrophin was confirmed using antibodies specific to regions of the dystrophin protein that are not expressed in revertant fibers. Approximately 93% of the fibers that were analyzed 24 weeks following transplantation of mdx 5cv SCs treated with PNA-COR C and that were identified using an antibody specific to the C- terminal region of the dystrophin protein were also positive for the region of the dystrophin protein encoded by exons 10 and 11 (Mandys-1011) and exon 32 (Mandys-18) (Supporting Information Fig. S6). Finally, correction of the genetic defect was confirmed in fibers that expressed dystrophin at the genomic level using direct sequencing of PCR products obtained from excised fibers that expressed dystrophin (Supporting Information Fig. S7). Taken together, our results clearly demonstrate that the loss of dystrophin expression typically seen using systems STEM CELLS