REPORTS

Cite as: C. Long et al., Science 10.1126/science.aad5725 (2015).

Duchenne muscular dystrophy (DMD) is a fatal muscle dis-ease affecting 1 in 3500 boys. Cardiomyopathy and heart failure are common, incurable and lethal consequences of DMD. The disease is caused by mutations in the gene encod-ing dystrophin (DMD), a large intracellular protein that links the dystroglycan complex at the cell surface with the underlying cytoskeleton, thereby maintaining integrity of muscle cell membranes during contraction (1, 2). In the ab-sence of dystrophin, muscles degenerate, causing weakness and myopathy (3). Many therapeutic approaches for DMD have failed, at least in part because of the size of the dystro-phin protein and the necessity for life-long restoration of dystrophin expression in the myriad skeletal muscles of the body as well as the heart.

The CRISPR (clustered regularly interspaced short pal-indromic repeats)/Cas9 (CRISPR-associated protein 9) sys-tem allows precise modification of the genome and represents a potential means of correcting disease-causing mutations (4, 5). In the presence of single guide RNAs (sgRNAs), Cas9 is directed to specific sites in the genome adjacent to a protospacer adjacent motif (PAM), causing a double strand break (DSB). When provided with an addi-tional DNA template, a precise genomic modification is generated by homology-directed repair (HDR), whereas in the absence of an exogenous template, variable indel muta-tions are created at the target site via non-homologous end-joining (NHEJ) (6). Previously, we used CRISPR/Cas9 to

correct a single nonsense mutation in Dmd by HDR in the germ line of mdx mice, which allowed the restoration of dystrophin protein expression (7). However, germ line ge-nomic editing is not feasible in humans (8) and HDR does not occur in post-mitotic adult tissues, such as heart and skeletal muscle (9), necessitating alternative strategies of gene correction in postnatal tissues. Here, we devised a method to correct Dmd mutations by CRISPR/Cas9-mediated NHEJ (termed “Myoediting”) in postnatal muscle tissues following delivery of gene editing components using adenovirus-associated virus-9 (AAV9), which displays high tropism for muscle (10, 11).

The dystrophin protein contains several domains (fig. S1), including an actin-binding domain at the N terminus, a central rod domain with a series of spectrin-like and actin-binding repeats, and WW and cysteine-rich domains at the C terminus that mediate binding to dystroglycan, dys-trobrevin and syntrophin (12). The actin-binding and cyste-ine-rich domains are essential for function, but many regions of the protein are dispensable (3). It has been esti-mated that as many as 80% of DMD patients could benefit from exon skipping strategies that bypass mutations in non-essential regions of the gene and partially restore dystro-phin expression (13). CRISPR/Cas9-mediated correction of DMD mutations in patients’ induced pluripotent stem cells (14) and immortalized myoblasts (15) has validated this ap-proach in vitro. Similarly, adenovirus-mediated gene editing

Postnatal genome editing partially restores dystrophin expression in a mouse model of muscular dystrophy Chengzu Long,1,2,3* Leonela Amoasii,1,2,3* Alex A. Mireault,1,2,3 John R. McAnally,1,2,3 Hui Li,1,2,3 Efrain Sanchez-Ortiz,1,2,3 Samadrita Bhattacharyya,1,2,3 John M. Shelton,4 Rhonda Bassel-Duby,1,2,3 Eric N. Olson1,2,3† 1Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA. 2Hamon Center for Regenerative Science and Medicine, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA. 3Sen. Paul D. Wellstone Muscular Dystrophy Cooperative Research Center, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA. 4Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.

*These authors contributed equally to this work.

†Corresponding author. E-mail: eric.olson@utsouthwestern.edu

CRISPR/Cas9-mediated genome editing holds clinical potential for treating genetic diseases, such as Duchenne muscular dystrophy (DMD), which is caused by mutations in the dystrophin gene. To correct DMD by skipping mutant dystrophin exons in postnatal muscle tissue in vivo, we used adeno-associated virus-9 (AAV9) to deliver gene editing components to postnatal mdx mice, a model of DMD. Different modes of AAV9 delivery were systematically tested, including intra-peritoneal at postnatal day (P) 1, intra-muscular at P12, and retro-orbital at P18. Each of these methods restored dystrophin protein expression in cardiac and skeletal muscle to varying degrees and expression increased from 3 to 12 weeks post-injection. Postnatal gene editing also enhanced skeletal muscle function, measured by grip strength tests 4 weeks post-injection. This method provides a potential means of correcting mutations responsible for DMD and other monogenic disorders after birth.

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was shown to restore dystrophin expression in specific mus-cles of mdx mice following intramuscular injection (16), but adenoviral delivery is less therapeutically favorable (17).

Shown in Fig. 1A is the strategy whereby CRISPR/Cas9-mediated NHEJ can create internal genomic deletions to bypass the premature termination codon in exon 23 respon-sible for the dystrophic phenotype of mdx mice, potentially allowing reconstitution of the Dmd open reading frame. In principle, this approach could be applied to many mutations within the gene. An advantage of this approach is that it does not require precise correction of the disease-causing mutation. Instead, imprecise deletions that prevent splicing of mutant exons are sufficient to restore dystrophin protein expression.

To test whether Myoediting could be adapted to skip the Dmd mutation in exon 23 in mdx mice, we first evaluated a pool of sgRNAs that potentially target the 5′ and 3′ ends of exon 23 (supplementary materials, fig. S2, and table S1). We co-injected Cas9 mRNA with sgRNA-mdx (directed toward the mutant sequence in exon 23) and either sgRNA-R3 or sgRNA-L8 (targeting the 3′ and 5′ end of exon 23, respec-tively) into mdx zygotes without a HDR template (fig. S3A). Strikingly, ~80% of progeny mice lacked exon 23 (termed mdx-∆Ex23) (fig. S3, B and C, and table S2), representing a significant increase in the efficiency of mdx editing com-pared to HDR (7). Genomic PCR products from the target sites of exon 23 and RT-PCR products of mdx-∆Ex23 mice were cloned and sequenced, confirming the skipping of exon 23 (fig. S3, D to F). As a result of skipping exon 23, the open reading frame of Dmd was restored, allowing dystrophin protein expression (fig. S4A). In mdx-∆Ex23 mice, serum creatine kinase (CK) levels, a measure of muscle membrane permeability, and grip-strength tests showed restoration of muscle function (fig. S4, B and C). Control mdx mice with-out treatment (-) and mdx mice with Myoediting (+) were tested for potential off-target effects of Myoediting with sgRNA-R3 (fig. S5). Ten potential genome-wide off-target sites (OT-01 to OT-10) were predicted by the CRISPR design tool (http://crispr.mit.edu/) (see the supplementary materi-als and table S4) (7). Only the target site Dmd R3 of My-oedited mdx mice showed cleavage bands in the T7E1 assay and no off-target effects were detected in the top ten poten-tial off-target sites (fig. S5).

To apply Myoediting to postnatal muscle tissues, we de-livered Cas9 and sgRNAs to muscle of mice using AAV9, which displays tropism to cardiac and skeletal muscle (10, 11). To generate AAV-guide RNAs, sgRNA-mdx and sgRNA-R3 were cloned into AAV-sgRNA vector containing a human U6 promoter and green fluorescent protein (GFP) (Fig. 1B). We generated AAV-Cas9 using a unique AAV-Cas9 vector (miniCMV-Cas9-shortPolyA) (18, 19), which employs a “mini”-CMV promoter/enhancer sequence to drive expres-

sion of the humanized SpCas9. Different modes of AAV9 delivery and variations in timing of expression were system-atically compared to identify the optimal method for Dmd Myoediting in postnatal mdx mice: (1) intra-muscular (IM) at P12, (2) retro-orbital (RO) at P18 and (3) intra-peritoneal (IP) at P1 (see the supplementary materials) shown in Fig. 1C.

Following IM injection of P12 mice with AAVs, muscle tissues were analyzed by immunostaining for dystrophin expression 3-weeks later (Fig. 1D and fig. S6A). Native GFP identified AAV-mediated gene expression in myofibers. Skeletal muscle from the IM-injected mice showed a mosaic pattern of dystrophin-positive fibers (Fig. 1D). The percent-age of dystrophin positive myofibers was calculated as a fraction of total estimated fibers. In the mdx mouse shown in the Fig. 1D, 7.7 ± 3.1% (SD) of myofibers in the tibialis anterior (TA) muscle expressed dystrophin 3-weeks post-IM injection. Rescue increased to an estimated 25.5 ± 2.9% (SD) of myofibers by 6-weeks post-IM-AAV (three male mdx mice per group) (fig. S6A). Hematoxylin and eosin (H&E) staining of muscle showed that histopathologic hallmarks of muscu-lar dystrophy, such as necrotic myofibers, were diminished in TA muscle at 6-weeks post-AAV delivery. Inflammatory cell invasion and centralized myofiber nuclei were minimal, contrasting greatly to uninjected control mdx TA (fig. S6B).

RO injection of AAV into the venous sinus of the mouse represents an alternative to tail vein injection for the sys-temic administration via blood circulation in young mice. Muscle tissues from mice following RO AAV injection (RO-AAV) at P18 were examined by RT-PCR (Fig. 1E). RT-PCR of RNA from Myoedited mdx mice showed that deletion of exon 23 (∆Ex23) allowed splicing from exon 22 to 24 (lower band) and the intensity of ∆Ex23 bands was increased from 4- to 12- weeks post-RO AAV injection. Sequencing of RT-PCR products of the ∆Ex23 band confirmed that exon 22 spliced to exon 24 (Fig. 1F). Muscle tissues were analyzed by immunohistochemistry (Fig. 2) and H&E staining (fig. S7). At 4-weeks post-RO-AAV injection in mdx mice, 2.5 ± 1.1% (SD) of myofibers were dystrophin positive, while 1.1 ± 0.3% (SD) of cardiomyocytes were dystrophin positive. Progres-sive improvement with age was also observed from 4- to 8- and 12-weeks post-RO-AAV. Rescue increased to an estimat-ed 6.1 ± 3.2% (SD) of myofibers in TA muscle, and 5.0 ± 2.1% (SD) of cardiomyocytes by 8-weeks post-RO-AAV. At 12-weeks post-injection, 4.6 ± 3.2% (SD) of myofibers were dys-trophin positive in TA muscle and 9.6 ± 3.9% (SD) of cardi-omyocytes were dystrophin positive. Western blot analysis confirmed the restoration of dystrophin expression in both heart and skeletal muscle (fig. S8).

Following IP injection of AAV editing components (fig. S9), dystrophin expression was rescued in 1.4 ± 1.2% (SD) of TA myofibers and 1.1 ± 1.1% (SD) of cardiomyocytes in treat-

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ed mdx mice after 4 weeks. Higher percent correction was observed in mdx-injected mice at 8-weeks post-IP-AAV in-jection with 1.8 ± 1.2% (SD) of dystrophin positive myofibers and 3.2 ± 2.4% (SD) dystrophin-positive cardiomyocytes. Grip strength testing (see the supplementary materials) showed a significant increase in strength of mdx mice at 4 weeks post-IP-AAV injection compared to uninjected mdx controls (Fig. 3).

Semi-quantitative immunohistochemistry was performed to quantify dystrophin expression levels in WT and AAV-injected mdx mice (see the supplementary materials). Rela-tive integrated density measures of sarcolemmal dystrophin immunostaining in TA myofibers, when normalized to lam-inin immunostaining showed dystrophin protein level 23.7 ± 11.6% (SD) that of WT when AAV was delivered IP, 27.7 ± 6.6% (SD) of WT when delivered RO, and 53.2 ± 18.5% (SD) of WT when AAV was delivered IM (figs. S10 and S12A). By the same means of measure, integrated density of dystro-phin in cardiomyocytes showed dystrophin protein level 52.4 ± 14.3% (SD) that of WT when AAV was delivered IP, 71.1 ± 21.0% (SD) of WT when delivered RO, and 69.7 ± 19.8% (SD) of WT when AAV was delivered IM (figs. S11 and S12B).

IM, RO and IP injection all provide transducing poten-tial in organs and muscle groups remote from the injection site, presumably through intra-vasculature circulation of AAV. Dystrophin expression in mdx mice was restored in vascular smooth muscle cells by all three modes of AAV de-livery, but most effectively by RO (fig. S13A). In contrast, no modes of AAV delivery were able to cross the blood-brain barrier to restore dystrophin expression in hippocampal CA1/CA2 regions of mdx mice (fig. S13, B and C). AAV transduction across the blood-brain barrier and subsequent restoration of brain dystrophin expression will likely require other methods (20–23). We also harvested sperm from AAV injected male mdx mice and tested gene editing by T7E1 assay. No cleavage bands were detected (fig. S13D), however, more sensitive methods, such as deep sequencing might be required to evaluate the risk of unexpected germline editing.

Our results show that AAV-mediated Myoediting can rescue the reading frame and expression of dystrophin in postnatal mdx mice. The efficiency of restoration of dystro-phin-positive myofibers increases with time, likely reflecting persistent expression of gene editing components. Exon skipping by NHEJ-mediated genomic editing allows for the permanent removal of the disease-causing mutation and was ~10-fold more efficient than gene correction by HDR (7). Myoediting by NHEJ does not require precise genetic modification. Instead, any types of indels that disrupt either a splice donor or acceptor sequence in a mutant exon result in exon skipping. It is noteworthy that the consensus se-quence for splice acceptors is NAG, corresponding to the

PAM sequence for Cas9 from S. pyogenes (NGG or NAG), so any exon can potentially be skipped by this approach.

It has been estimated that even low level expression of dystrophin (4~15%) can partially ameliorate cardiomyopa-thy (24) and protect against eccentric contraction-induced injury in skeletal muscle (25). The efficiency of restoration of dystrophin expression observed following delivery of My-oediting components to mdx mice by AAV is therefore with-in the range expected to provide therapeutic benefit.

Off-target effects are a safety concern in the eventual translation of gene editing methods to humans. We did not observe off-target mutations at ten potential off-target sites in the mouse genome nor any abnormalities in mice follow-ing AAV9 delivery of Myoediting components. However, off-target mutations may occur at sites beyond those predicted in silico; hence a comprehensive and unbiased analysis, such as whole genome sequencing, would be an essential component of future efforts to establish the safety of this approach (26–28). Given that Myoediting offers the poten-tial for durable and progressive therapeutic response in post-mitotic adult tissue, we propose that this methodology may warrant investigation as a way to restore muscle func-tion in DMD patients, alone or in combination with other therapies (3, 29).

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ACKNOWLEDGMENTS

We thank D. Grimm and F. Schmidt for AAV-hCas9 and the sgRNA Cloning Vector plasmids and C. Wang for AAV9 packaging. We thank J. Schneider and P. Mammen for discussions, and C. Rodriguez for technical help; Z. Wang for input; S. Rovinsky and E. Plautz (UT Southwestern Neuro-Models Facility) for grip strength testing, X. Li and R. Gordillo (UT Southwestern Mouse Metabolic Phenotyping Core Facility) for CK level measurement, J. Cabrera for graphics. This work was supported by grants from the NIH (HL-077439, HL-111665, HL-093039, DK-099653, U01-HL-100401 and U54 HD 087351), Fondation Leducq Networks of Excellence, and the Robert A. Welch Foundation (grant 1-0025 to E.N.O.). The pSpCas9(BB)-2A-GFP (PX458) plasmid is available for purchase from Addgene under a material transfer agreement. The University of Texas Southwestern Medical Center and the authors (ENO, RBD, JMS, CL, JRM) have filed a patent application (#14/823,563) related to the use of CRISPR/Cas9 technology to treat muscle disease.

SUPPLEMENTARY MATERIALS www.sciencemag.org/cgi/content/full/science.aad5725/DC1 Materials and Methods Figs. S1 to S13 Tables S1 to S4 References (30–33) 2 October 2015; accepted 4 December 2015 Published online 31 December 2015 10.1126/science.aad5725

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Fig. 1. Permanent exon skipping in postnatal mdx mice by AAV-mediated Myoediting. (A) Strategy for bypassing exon 23 of the mdx locus by NHEJ. (B) AAV vectors for expression of Cas9 (AAV-Cas9, upper), guide RNAs and GFP (AAV-sgRNA, lower). ITR, inverted terminal repeat. RSV, Rous sarcoma virus promoter. U6, human U6 promoter (C) Different modes of AAV9 delivery. Black arrows indicate the post-injection time points for tissue collection. (D) Rescue of dystrophin expression in mdx mouse by IM injection of Myoediting components. Green fluorescent protein (GFP) and dystrophin immunostaining from serial sections of mdx mouse TA muscle is shown 3- and 6-weeks post-IM-AAV of AAV-Cas9/sgRNAs at P12 (three male mdx mice in each group). Asterisks indicate serial section myofiber alignment. Scale bar, 40 μm. (E) RT-PCR of RNA from Myoedited mdx mice indicates deletion of exon 23 (termed ∆Ex23, lower band) and shows increase in intensity of ∆Ex23 bands from 4 to 12 weeks post-RO injection (four male mdx mice in each group). Asterisk indicates the RT-PCR products with small deletions. M denotes size marker lane. bp indicates the length of the marker bands. (F) Sequence of the RT-PCR products of ∆Ex23 band confirmed that exon 22 spliced directly to exon 24, excluding exon 23.

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Fig. 2. Rescue of dystrophin expression in postnatal mdx mice by retro-orbital injection of AAV-Cas9/sgRNAs. (A) Dystrophin immunostaining of TA muscle is shown for WT, mdx and RO-AAV treated mdx mice at 4-, 8- and 12-weeks post-injection (RO-AAV at P18, four male mdx mice in each group). TA muscle of unedited mdx control mice exhibits myonecrosis, indicated by cytoplasm-filling autofluorescence (highlighted with white asterisks). (B) Dystrophin immunostaining of the heart is illustrated for WT, mdx, and RO-AAV treated mdx mice at 4-, 8- and 12-weeks post-injection (RO-AAV at P18, four male mdx mice in each group). Arrowheads indicate dystrophin positive cardiomyocytes in 4-weeks post-RO-AAV treated mdx mouse heart. Scale bar, 40 μm.

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Fig. 3. Forelimb grip strength of mdx, mdx-AAV-IP and WT mice at 4 weeks post-injection. mdx, mdx-AAV-IP and WT mice were subjected to grip strength testing to measure muscle performance (grams of force), and the mdx-AAV-IP mice showed enhanced muscle performance compared to mdx mice at 4 weeks of age. (mdx male control 34.7 ± 1.8%; mdx-AAV-injected male mice 48.4 ± 2.5%, WT male 71.8 ± 1.9% and mdx female control 29.7 ± 1.4%; mdx-AAV-injected female mice 45.5 ± 1.4%, WT female 75 ± 2.4%). Numbers of mice in each group are labeled in the bar, 6 trials for each mouse. Data are represented as mean ± SEM. Significant differences between conditions are indicated by asterisk (***P<0.0005).

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dystrophyPostnatal genome editing partially restores dystrophin expression in a mouse model of muscular

M. Shelton, Rhonda Bassel-Duby and Eric N. OlsonChengzu Long, Leonela Amoasii, Alex A. Mireault, John R. McAnally, Hui Li, Efrain Sanchez-Ortiz, Samadrita Bhattacharyya, John

published online December 31, 2015

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REFERENCES

http://science.sciencemag.org/content/early/2015/12/29/science.aad5725#BIBLThis article cites 33 articles, 7 of which you can access for free

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