Pesquisas que serão apresentadas no Congresso da Sociedade Americana de Terapia Celular e Gênica, maio de 2015

1) Efficient In Vitro Expansion and Gene Correction of Dystrophic Mouse Muscle Stem Cells

Mohammadsharif Tabebordbar, Jason Cheng, Amy Wagers. Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA; Howard Hughes Medical Institute, Chevy Chase; Harvard Stem Cell Institute, Cambridge, MA

Duchenne Muscular Dystrophy (DMD) is a progressive muscle wasting disease caused by mutations in the DYSTROPHIN gene. Gene correction and autologous transplantation of muscle stem cells (i.e., satellite cells) presents a promising therapeutic approach for restoring DYSTROPHIN expression to muscle fibers in these patients; however the low frequency of satellite cells in adult muscle is an obstacle for isolating sufficient numbers of cells for engraftment. To address this issue, we have sought culture conditions that would support the ex-vivo expansion of satellite cells and potentially provide more cells for transplantation. We found that forskolin, an adenylyl cyclase activator, dramatically expanded mouse satellite cells in culture. Forskolin-treated cultured cells retained the immunophenotypic characteristics of engraftable satellite cells, and transplantation of compound-treated cells into dystrophic muscle yielded a significantly higher level of engraftment compared to control cells. Forskolin also dramatically expands satellite cells from mdx mouse model of DMD in culture. As a proof of concept for a combined gene and cell therapy approach for treating DMD, we targeted the mutated Dystrophin locus in ex-vivo expanded forskolin treated dystrophic satellite cells using CRISPR/Cas9 gene editing technology and enriched for the targeted cells using an endogenous fluorescent reporter system. Expansion and gene correction of muscle satellite cells in culture as described here provides the possibility of improving combined gene and cell-based therapies for neuromuscular disorders.

 2) Precise Control of CRISPR-Cas9 Mediated Gene Editing for Correcting Mutation of Duchenne Muscular Dystrophy in iPS Cells

Hongmei L. Li, Norko Sasakawa, Kentaro Ishida, Peter Gee, Akitsu Hotta. Department of Reprogramming Science, Center for iPS Cell Research & Application (CiRA), Kyoto University, Kyoto, Japan

Duchenne Muscular Dystrophy (DMD) is one of the most common and severe inherited neuromuscular disease caused by the loss-of-function mutations in Dystrophin gene on X chromosome. Currently, no effective treatment available for DMD, including gene augmentation therapy, since large size of the Dystrophin gene hamper the delivery by viral vectors. Exon skipping to modulate mRNA splicing patterns using antisense oligonucleotide is a promising approach currently investigated in clinical trials, however, the effect of antisense oligos is transient. 
Recent progress on targeted gene editing by engineered nucleases, such as TALENs or CRISPR/Cas9, have evolutionally broaden our ability to precisely modify the genomic sequence at desired locus, including genetic correction of Dystrophin gene. At the same time, however, undesired mutagenesis at the off-target loci with a few base-pair mismatches is a big concern. 
To this end, we previously developed a method to identify “unique sequence” in the human genome by using a database of short and unique k-mer sequences [Li HL et al., Stem Cell Reports, 4(1):143-54, 2015]. Our “uniqueness” data is useful to design a target site for engineered nucleases with high sequence specificity and minimum number of putative off-target sites, and the data is publically available at
Moreover, to precisely control the genomic cleavage activity of CRISPR-Cas9, we constructed doxycycline inducible expression vector and Cas9 protein fused a nuclear receptor to mediate nuclear shuttling upon addition of receptor substrate. We show that both transcriptional and posttranscriptional regulations of CRISPR-Cas9 protein are effective to modulate DNA cleavage activity. 
To test the effectiveness of CRISPR system to correct disease mutation, we took advantages of patient-derived induced pluripotent stem cells (iPSCs) as a platform for testing various gene correction approaches. As a disease modeling for DMD gene thrapy, we derived integration-free iPS cell lines from a DMD patient who lacks the exon 44 in the Dystrophin gene, which is one of the most comon deletions of single exon. We will evaluate the dual-controlable Cas9 approache in patient-derived iPSCs, since gene-corrected iPSCs hold a promise to serve as a cell source for future DMD therapy.

3) A Novel Gene Editing-Based Strategy for Myotonic Dystrophy Type 1

Mirella Lo Scrudato, Samia Martin, Geneviève Gourdon, Denis Furling, Ana Buj-Bello. Genethon, INSERM UMR 951, Evry, France; INSERM UMR 1163, Institut Imagine, Université Paris Descartes-Sorbonne Paris Cité, Paris, France; UPMC-AIM UMR S974, INSERM U974, CNRS FRE 3617, Institut de Myologie, Université Pierre et Marie Curie Paris 6, Paris, France

Myotonic dystrophy type 1 (DM1) is an autosomal dominant muscular disease caused by an expansion of the CTG trinucleotide repeat located in the 3' untranslated region (3′ -UTR) of the DMPK gene. The size of the expansion correlates with disease severity and to date there is no effective treatment for patients. Mutated DMPK transcripts aberrantly interact with proteins in the nucleus and form stable complexes where splicing factors are sequestered. As a consequence, the alternative splicing of numerous transcripts is pathogenically misregulated. 
We aim at developing a strategy to delete the CTG expansion from the human DMPK locus by using the CRISPR/Cas9 system. We have generated several constructs which express a small size Cas9 nuclease and guide-RNAs (gRNAs), and show their ability to excise the targeted DNA region in various cell lines. We are currently producing adeno-associated vectors for in vivo delivery of these constructs in a DM1 mouse model.

4) Translating DUX4-Targeted RNAi Therapy for FSHD

Lindsay M. Wallace, Jacqueline S. Domire, Scott Q. Harper. Center for Gene Therapy, The Research Institute at Nationwide Children's Hospital, Columbus, OH; Department of Pediatrics, The Ohio State University, Columbus, OH

OBJECTIVE: Develop a DUX4-targeted RNAi-based gene therapy for Facioscapulohumeral muscular dystrophy (FSHD)

BACKGROUND: FSHD, the third most prevalent muscular dystrophy, is an autosomal dominant disorder that most commonly causes progressive weakness in muscles of the face, shoulder girdle, and limbs. FSHD was formally classified as a major form of muscular dystrophy in 1954, but the pathogenic events leading to the disease have only recently started coming into focus. Several studies now support an FSHD model involving aberrant expression of the DUX4 gene, which encodes a myotoxic transcription factor. The emergence of DUX4 represented a momentum shift in the FSHD field as it provided an important target for therapy design. As FSHD is currently untreatable, developing effective therapies is a critical need in the field. We hypothesized that an FSHD treatment should center on inhibiting DUX4 expression and/or activity, and have implemented several strategies to accomplish this goal. In the first published study on DUX4 inhibition, we demonstrated that AAV-mediated RNAi gene therapy could effectively suppress DUX4 expression and protect mouse muscles from its toxic effects. Specifically, our therapeutic DUX4-targeted artificial microRNAs improved histological, molecular, and functional outcomes associated with DUX4 expression in muscle. We are now working to translate this therapy through optimization of vector and delivery route. In addition, as over-expression of inhibitory RNAs have been associated with off-target effects, we are assessing the safety of this approach using dose-escalation and toxicology studies. The latter experiment is particularly important since the potential toxicity of RNAi therapy has not been previously assessed in muscles.

DESIGN/METHODS: We addressed 4 issues in this study, as we proceed down a path toward translation: (1) removed the GFP reporter from our 1st-generation AAV vectors; (2) tested additional DUX4-targeted miRNA sequences; (3) developed muscle-restrictive promoters for miRNA expression; and (4) determined the safety of muscle-targeted miRNA therapy through dose-escalation toxicology studies, using direct intramuscular injection and vascular delivery.

RESULTS: Removal of the GFP reporter gene had no deleterious effect on therapeutic efficacy and positioned our new vectors for potential clinical usage. Our alternative DUX4 miRNA proved to be equally effective at preventing DUX4-induced toxicity as the original sequence, and we have established safety thresholds by determining the maximum tolerable AAV.miRNA dose in mice for IM and IV delivery. At very high levels, all AAV-delivered miRNAs showed some toxicity, but importantly we found no deleterious overt pathology related to miRNA treatment at vector doses proven effective for DUX4 suppression in mice, over several timepoints. This toxicology data is promising for translating this strategy, especially considering DUX4 is markedly less abundant in human FSHD muscle than in our mouse model (which expresses the gene at very high levels).

CONCLUSIONS: This study provides important data necessary for translating an RNAi-based therapy for FSHD.

5(Exploiting Retroviral Recombination for the Delivery of Full-Length Dystrophin cDNA

John R. Counsell, Zeinab Asgarian, Jinhong Meng, Veronica Ferrer, Steven J. Howe, Francesco Muntoni, Jennifer E. Morgan, Olivier Danos. Dubowitz Neuromuscular Centre, UCL Institute of Child Health, London, United Kingdom; Wolfson Gene Therapy Unit, UCL Cancer Institute, London, United Kingdom; Molecular and Cellular Immunology, UCL Institute of Child Health, London, United Kingdom

Duchenne muscular dystrophy (DMD) is caused by loss of dystrophin expression in patient muscle cells. Restoration of dystrophin expression could alleviate the symptoms of DMD, although the large size of full-length dystrophin cDNA (11.1kb) and the restricted packaging capacity of viral vectors limit the application of standard gene transfer methods.
Retroviral vectors have a pseudo-diploid RNA genome which can co-package two distinct RNA genomes to form a heterozygous virion. Template-switching between co-packaged strands is a common event during HIV-1 reverse transcription, which can lead to recombination and the production of chimeric proviruses. Template-switching can be influenced by a number of cis and trans-acting factors such as structures in the viral packaging sequences and mutations of reverse transcriptase.
In this work, the recombinogenic characteristics of HIV-1 based lentiviral vectors have been exploited for the purpose of reconstituting full-length dystrophin cDNA from overlapping 5' and 3' portions of the full-length coding sequence. The vector has been initially optimised using a small bicistronic cassette to evaluate the recombination efficiency in standard lentiviral vectors compared with those harbouring mutations in the HIV-1 packaging sequence and reverse transcriptase.
Interestingly, the results suggested that a standard lentivirus vector could deliver a full-length, recombined provirus with greater efficiency than vectors harbouring mutations to cis and trans factors. A multiplicity of infection (MOI) of 40 heterozygous vector particles per target cell was sufficient to obtain a reconstituted full-length Neomycin-IRES-GFP provirus in 40% of transduced HeLa cells. When the viral RNA components were further modified to render functional provirus synthesis dependent on recombination (schematic displayed in figure 1), the vector was capable of reconstituting a full-length dystrophin-gfp fusion protein in HEK293t cells. Flow cytometry analysis showed that heterozygous vectors produced 0.1% GFP-positive cells following a MOI50 vector dose. Furthermore, genomic DNA extracted from these cells contained a full-length dystrophin provirus, which was detected by nested PCR and sequenced by clonal analysis.
This work reveals that retroviral recombination offers a means to circumvent retroviral packaging limitations and may be of use in dystrophin gene therapy following further optimisation.

6) Eteplirsen, a Phosphorodiamidate Morpholino Oligomer (PMO) for the Treatment of Duchenne Muscular Dystrophy (DMD): 168 Week Update on Six-Minute Walk Test (6MWT), Pulmonary Function Testing (PFT), and Safety

Jerry R. Mendell, Louise Rodino-Klapac, Zarife Sahenk, Kandice Rouch, Lauren Bird, Linda Lowes, Lindsay Alfano, Katherine Berry, Sarah Lewis, Kim Shontz, Kevin Flanigan, Christopher Shilling, Petra Duda, Jay Saoud. The Research Institute, Nationwide Children's Hospital, Columbus, OH; Sarepta Therapeutics, Cambridge, MA


DMD is a rare, degenerative, X-linked recessive genetic disease that results in progressive muscle loss and premature death. DMD is caused by mutations in the dystrophin gene that lead to a reading frame shift and premature translation termination. Exon skipping, a promising disease-modifying approach for DMD, can be induced by eteplirsen, a charge neutral PMO that selectively binds to exon 51 of dystrophin pre-mRNA, restoring the open reading frame and enabling production of an internally truncated yet functional dystrophin protein as found in the less severe dystrophinopathy, Becker muscular dystrophy (BMD). 


Twelve eligible boys, aged 7-13 years were randomized 1:1:1 to eteplirsen 30 or 50 mg/kg/wk, or placebo for 24 weeks. All patients transitioned into the ongoing open-label extension trial at Week 25 taking eteplirsen 30 or 50 mg/kg. Initial placebo-treated were denoted “placebo-delayed” after starting the PMO. Efficacy endpoints included 6MWT, PFT, and %-dystrophin positive fibers. Safety assessments included AE recording, ECG, ECHO, and safety laboratory testing. 


After more than 3 years of treatment, all patients previously evaluable on 6MWT (mITT; n=10) showed continued ambulation. The boys on continuous eteplirsen from Week 1 (n=6) declined 76.7 meters in walking ability through week 168. This was 65 meters better than placebo-delayed (n= 4; p≤0.017), starting eteplirsen at Week 25. Of particular interest, the placebo-delayed declined 68 meters through Week 36 and then showed a course almost identical to the eteplirsen cohort (placebo-delayed declined 73 meters; continuous eteplirsen declined 76 meters). This is most likely related to the time needed for the production of meaningful levels of dystrophin (∼12 weeks post-treatment). All patients, even those age 12-15, continue to demonstrate an ability to climb stairs, to rise from supine, and to raise a hand to the mouth at a rate higher than what was observed in a natural history study of glucocorticoid-naïve or steroid-treated boys with DMD (Henricson et al., 2013). All 12 patients, including the two non-ambulatory patients, demonstrated PFT stability from baseline through Week 168, including MIP (+11.1%, p=NS), MEP (+13.5%, p=NS), and MIP/MEP %-predicted (-2.4%/-6.3%, p=NS).

No deaths, discontinuations, treatment-related SAEs, immune activation including infusion reactions, or clinically significant abnormal laboratory, ECG, or ECHO findings were reported. 


Eteplirsen treated patients demonstrated a significant clinical benefit on the 6MWT over 168 weeks. The observed decline in walking distance contrasts with the decline observed in DMD natural history of comparative age. Eteplirsen was well tolerated, with no clinically significant treatment related adverse events.

7)Intramuscular and Systemic Induction of the N-Truncated Dystrophin By Out-Of-Frame Exon 2 Skipping Restores Muscle Function in the Dup2 Mouse, Providing Further Support for a Therapeutic Pathway for 5' DMD Mutations

Nicolas Wein, Adeline Vulin, Tabatha Simmons, Felecia Gumienny, Nianyuan Huang, Francesco Muntoni, James Ervasti, Robert Weiss, Kevin Flanigan. Center for Gene Therapy, Nationwidechildrens Hospital, Columbus, OH; Developmental Neuroscience, UCL Institute of Child Health, London, United Kingdom; Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN; Department of Human Genetics, The University of Utah School of Medicine, Salt Lake City, UT

Most mutations that truncate the reading frame of the DMD gene result in loss of dystrophin expression and lead the severe Duchenne muscular dystrophy. However, frame-truncating mutations within the first five exons of DMD result in mild dystrophinopathy with expression of a N-truncated dystrophin. We have recently shown that this is due to activation of an internal ribosome entry site (IRES) within exon 5 resulting in translation from an exon 6 AUG codon. We demonstrated that this IRES is active in patients expressing the N-truncated dystrophin, raising the possibility of the therapeutic use of this isoform. To explore this we developed a novel out-of-frame exon-skipping approach that uses AAV-mediated U7snRNA to efficiently skip exon 2. By injecting this AAV vector into a DMD mouse model carrying a duplication of exon 2 (Dup2), this generates a truncated reading frame, leading to activation of the IRES and synthesis of the N-truncated isoform. 

We now demonstrate that despite lacking the first half of the canonical actin binding domain 1, this N-truncated protein is highly functional. Intramuscular injection of the AAV1.U7snRNA vector into Dup2 mice results in high levels of expression of the N-truncated isoform by 4 to 6 weeks post-injection, along with complete correction of the physiologic and pathologic features as measured by Evans blue dye uptake, hindlimb grip strength, tibialis anterior specific force, and force correction after eccentric contraction. Preliminary results supports that systemic delivery of AAV9.U7snRNA vector into Dup2 mice induce expression of this functional isoform into all muscle including heart and diaphragm, thereby improving muscle histopathology. 

Following treatment, a genome-wide normalized RPF-Seq data analysis (Ribosome Protected Fragment) was performed to check if the treatment restored the Haslett gene lists (gene altered in DMD) to a 'non-dystrophic' pattern. Our data clearly indicates that the treatment restored the global expression pattern to a more normal pattern. This level of correction to that of control mice supports the idea that this novel therapeutic approach should be beneficial for the 6% of patients with mutations within the first five exons of DMD.

8) Targeted Genome Editing in Spinal Muscular Atrophy

Annalisa Lattanzi, Matteo Bovolenta, Samia Martin, Vincent Mouly, Fulvio Mavilio. Genethon, Evry, France; Department of Life Sciences and Biotechnology, University of Ferrara, Ferrara, Italy; Institut de Myologie, GH Pitié-Salpétrière, Paris, France

Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder caused by mutations in the survival-motor-neuron 1 (SMN1) telomeric gene. Deficiencies in the ubiquitous SMN function affect multiple tissues and organs; however neuronal tissue is primarily sensitive, resulting in α-motor neuron degeneration in the ventral horn of the spinal cord with subsequent neuromuscular-junction dysfunction and proximal muscle weakness. The onset of disease and degree of severity are variable in patients and they are determined in part by multiple copies of the centromeric homologue SMN2 that inversely correlate with the phenotypic severity. Indeed, SMN2 gene mainly produces a truncated form SMNΔ7 by aberrant alternative splicing and a small amount (∼10%) of the fully active full-length SMN, thus buffering the SMN deficiency. A potential strategy for treating SMA patients is to increase SMN levels in the affected tissues, hence gene therapy and modifiers of SMN2-alternative splicing have proved therapeutic efficacy in SMA animal models.
In this study, we explored the possibility of applying targeted genome editing technology to the human SMN locus in order to revert the SMN2 sequence to a SMN1-like sequence that may undergo proper splicing under the the endogenous transcriptional control. The resulting correction would be permanent and lead to longlasting protein production in gene-edited cells. We used the streptococcus pyogenes Cas9-CRISPR system to target the SMN2 gene at different locations. Two main strategies were explored: i) SMN1_exon7 addition/correction by promoting homology-driven DNA repair, ii) SMN2_intron7_ intronic-splicing-silencer (ISS-N1) mutation and correction of SMN2 aberrant splicing, by exploiting the non-homologous end-joining (NHEJ) pathway. Plasmids encoding Cas9-GFP under the control of CMV promoter, and selected gRNAs downstream to the Pol-III U6 promoter (Addgene) were transfected in HEK-293T cell line and in immortalized myoblasts derived from either healthy donors or SMA patients. Transfection efficiency was estimated as percentage of GFP-expressing cells (20-50% and 1-10%, respectively) and nuclease activity detected by Surveyor assay and target site sequencing. In particular, in SMA patient-derived myoblasts we detected mutations (indels) at the level of the induced DNA double-strand break at ∼30% frequency. Levels of SMN restoration will be investigated by qPCR of the different species of SMN transcripts and by western blotting of SMN protein. The goal of this study is to provide an in vitro proof of principle of effective gene correction in SMA patient-derived cells. In the context of a multisystemic, complicated disease such as SMA, targeted genome editing strategy could represent an additional therapeutic tool.

9) First-in-Human Study of NS-065/NCNP-01; the Morpholino Based Antisense Oligonucleotide for Exon 53 Skipping in Duchenne Muscular Dystrophy

Takashi Saito, Tetsuya Nagata, Satoru Masuda, Maiko Suzuki, Harumasa Nakamura, Hirofumi Komaki, Shin'ichi Takeda. Department of Molecular Therapy, National Institute of Neuroscience, National Center of Neurology and Psychiatry (NCNP), Kodaira, Tokyo, Japan; Translational Medical Center, NCNP, Kodaira, Tokyo, Japan; Department of Child Neurology, NCNP, Kodaira, Tokyo, Japan

Currently, phase 2/3 clinical trials of exon 51 skipping for Duchenne Muscular Dystrophy (DMD) are being conducted and have been shown promising results for NDA. Next to the patients treatable by exon 51 skipping, the patients amenable to exon 53 skipping are the second largest population; therefore a development of exon 53 skipping drug has high priority. National Center of Neurology and Psychiatry, with Kyoto-based pharmaceutical company Nippon Shinyaku, had jointly developed the exon 53 skipping drug since 2009 and started an investigator-initiated clinical trial from June 2013 (UMIN Clinical Trial ID: UMIN000010964, NCT02081625). The NS-065/NCNP-01 is a morpholino based antisense oligonucleotide that has been developed to skip the exon 53 of the dystrophin gene and to treat DMD patient amenable to the exon 53 skipping. It has been confirmed potent efficacy and high safety in pre-clinical studies. This clinical trial is an exploratory phase 1, single-site, first-in-human study. The primary endpoints are the safety and tolerability and the secondary endpoints are the pharmacokinetics and efficacies (dystrophin recovery). Three doses cohort design (1.25 mg/kg, 5 mg/kg and 20 mg/kg) was adopted and all subjects were dosed weekly intravenous infusion for 12 weeks. Mainly non-ambulant subjects were recruited because the dose and duration in this trial was not enough to expect functional improvement. Of total 10 subjects, each three subjects were randomly assigned to 1.25 mg/kg or 5 mg/kg cohorts, and four subjects were assigned to 20 mg/kg cohort. An in vitro confirmation of dystrophin recovery and exon 53 skipping in subject-derived cells was one of inclusion criteria. Subject's mutations were classified into any of exons 45-52, 48-52 or 49-52 deletion; all NS-065/NCNP-01 treated cells satisfied the criteria. One week after an initial dosing for the first subject in each cohort, next subjects were dosed. Safety review committee advised the principal investigator whether or not to proceed to next cohort. At the end of 2014, dosing to all subjects had completed; no serious adverse events were observed. Dystrophin expression is to be evaluated by Western blotting and immunofluorescent staining. The analysis is still ongoing, but an immunofluorescent image analysis will be performed as objective and quantitative as possible by automated measurements. We would report progress of the first-in-human study of NS-065/NCNP-01; the morpholino based exon 53 skipping drug for DMD.

10) Off-Target Analysis of a rAAV-U7snRNA Vector Used for the Treatment of Duchenne Patients By Exon Skipping

Claire Domenger, Aurélie Lardenois, Marine Allais, Virginie François, Marie Montus, Laurent Servais, Thomas Voit, Philippe Moullier, Caroline Le Guiner. Atlantic Gene Therapies, INSERM UMR 1089, Université de Nantes, CHU de Nantes, Nantes, France; Atlantic Gene Therapies, INRA UMR 703, ONIRIS, Nantes, France; Généthon, Evry, France; Institut de Myologie, UPMC-INSERM UMR 974, CNRS FRE 3617, Paris, France; Department of Molecular Genetics and Microbiology, University of Florida, Gainesville

Duchenne muscular dystrophy (DMD) is a severe muscle wasting disorder affecting 1 male in 5000 and caused by mutations in the gene encoding for the Dystrophin protein. So far, there are no strongly effective treatments but gene-based therapies are currently being developed. Among them, the selective removal by exon skipping of exons flanking an out-of frame mutation in the dystrophin messenger can result in shorter in-frame transcripts that are translated into functionally active Dystrophin. Exon skipping can be achieved using modified U7-small nuclear RNA (snRNA) in which a therapeutic antisense oligonucleotide (AO) is cloned. Such optimized U7snRNA, called U7.OPT, can be packaged in recombinant vectors derived from Adeno-Associated Virus (rAAV), allowing a long-lasting repair of the dystrophin messenger after one single administration. 
We recently published the high ability of a rAAV8.U7.OPT vector to transduce the skeletal muscle but also the liver and restore the open reading frame of the dystrophin transcript in GRMD dogs muscles injected in one forelimb. We are now moving forward to a phase I/II clinical trial in DMD patients treatable by skipping of the exon 53 of the dystrophin messenger. 
Because an abundant presence of the therapeutic U7.OPT RNA can in theory lead to partial hybridization to non-targeted messenger(s) with unwanted side effects, we looked for possible off-target events. Primary human myotubes and hepatocytes, untransduced or transduced with our clinical rAAV.U7.OPT vector, served as our in vitro model system. The skipping of exon 53 of the human dystrophin messenger upon vector transduction was validated. In silico analysis using the miRanda algorithm identified some gene candidates expressed in liver and muscle, i.e. messengers which could be the target of our dystrophin-specific AO. Three of them were analyzed by RT-PCR and RTqPCR and showed not detectable exon skipping events or differential expression. Other candidates are currently analyzed. Simultaneously, further investigation included a full transcriptomic analysis by RNA sequencing comparing the samples obtained from untransduced vs transduced primary cells. Preliminary results revealed differential gene expression patterns between the two types of samples. We are now exploring the respective impact of the rAAV transduction itself vs the AO effect per se to finally identity potential off-target events. 
The complete data will be presented and discussed in the context of our future clinical trial in DMD patients.

11) Screening and Optimization of Antisense Oligonucleotide for Skipping Human Dystrophin Exon 51 and 53

Bo Wu, Peijuan Lu, Sapana N. Shah, Stephanie Milazi, Lauren E. Bollinger, Qi Long Lu. McColl Lockwood Laboratory for Muscular Dystrophy Research, Carolinas Medical Center, Charlotte, NC

Duchenne muscular dystrophy (DMD) results from dystrophin gene mutations, causing shift of the reading frame and preventing production of a functional protein. Most DMD mutations occur in the parts of the gene that are not critical for its function, therefore restoration of the reading frame by antisense oligonucleotide-mediated exon skipping is a viable approach. The efficacy of antisense therapy has now been proven in animal models and in clinical trials. 

Therapeutic effect of exon skipping largely depends on the efficiency of individual antisense oligoneotide, which has to be identified by screening. This study aims specifically to search for the most effective morpholino (PMO) oligomer to target the human dystrophin exon 51 and exon 53 for the correction of the relevant DMD mutations. We established the GFP reporter myoblast cell cultures and screened more than 40 PMOs targeting each of exon 51 and exon 53. We also examined the PMOs in normal human myoblast cultures and in humanized DMD (hDMD) mice with local delivery by i.m injection to identify PMOs of maximal skipping potency. Finally we selected 5 oligomers as vivo-PMOs targeting each of the exon 51 and 53 and examined their exon skipping efficiency in the hDMD mice by systemic delivery. We were able to identify PMOs with high exon skipping efficiency in all muscles. The selected PMOs will be further validated in patient-derived fibroblasts and then be applied to clinical trials for DMD treatment.

12)  Spell Checking Nature: A Therapeutic Use of the CRISPR/Cas9 System in Duchenne Muscular Dystrophy

Daria Wojtal, Dwi U. Kemaladewi, Zeenat Malam, Sarah Abdullah, Elzbieta Hyatt, Victoria Huang, Vincent Mouly, Thomas Voit, Francesco Muntoni, Evgueni A. Ivakine, Ronald D. Cohn. Genetics and Genome Biology Program, Research Institute of The Hospital for Sick Children, Toronto, ON, Canada; Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada; Institut de Myologie, Université Pierre et Marie Curie-Paris 06 UM76, INSERM U974, CNRS UMR 7215, Paris, France; Dubowitz Neuromuscular Centre, Institute of Child Health and Great Ormond Street Hospital, London, United Kingdom; Division of Clinical and Metabolic Genetics; Paediatrics and Molecular Genetics, The Hospital for Sick Children, Toronto, ON, Canada

Recently the high precision genome-modifying technology, originated from a prokaryotic defense mechanism called the Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) system, has been harnessed as a tool for programmable, high precision gene modification in eukaryotes. Thus far it has been shown to be highly effective in inactivating genes, however little is known about the gene editing and regulation capabilities of this system. As a proof of concept we utilized CRISPR/Cas9 system to study three different therapeutic approaches for Duchenne Muscular Dystrophy (DMD), a devastating X-linked neuromuscular disorder. 

The first approach is aimed at approximately 26% of patients that have point mutations in DMD gene. Here we used gene editing to correct DMD E2035X, a pathogenic mutation in DMD patient myoblasts by designing short RNA sequences complementary to the locus called guides, paired with the Streptococcus pyogenes Cas9. We were able to generate targeted double stranded DNA breaks that were corrected through homology directed repair using single stranded oligos carrying the corrected DNA sequence. Other pathogenic DMD mutations are duplications, which are found in approximately 13% of DMD patients. Utilizing a modified CRISPR strategy that involves a double nuclease approach we deleted a 145 kb (Exons 18-30) duplication and restored dystrophin expression in patient cells. Furthermore, as an universal, mutation-independent treatment approach, we have also utilized a third modified CRISPR system to upregulate utrophin expression to compensate for the loss of dystrophin. In DMD patient myoblasts, we utilized S.pyogenes Cas9, fused to transcriptional activator VP160 to target utrophin promoter. We demonstrated 2.5-5 fold utrophin upregulation resulting in increased expression of β-dystroglycan, providing evidence for functional significance of this strategy. 

Lastly, in order to determine if this tool is of potential clinical significance, we have began to interrogate CRISPR/Cas9 system in dystrophin-deficient mdx mouse. We have developed strategies for the delivery of CRISPR machinery via rAAV with the goal to achieve dystrophin editing and utrophin upregulation simultaneously in vivo. Taken together, our proof of concept studies provide insights into the potentially far-reaching impact of the therapeutic benefits of the CRISPR system that can be adapted to target a variety of mutations in DMD.

13) Correction of the Dystrophin Gene By the CRISPR/Cas9 System in a Mouse Model of Muscular Dystrophy

Christopher E. Nelson, David G. Ousterout, Jacqueline N. Robinson-Hamm, Eirik A. Moreb, Ruth M. Castellanos, Fei Ann Ran, Winston Yan, Feng Zhang, Aravind Asokan, Charles A. Gersbach. Duke University, Durham, NC; Duke University Medical Center, Durham, NC; Broad Institute of MIT and Harvard, Cambridge, MA; MIT, Cambridge, MA; University of North Carolina, Chapel Hill, NC

Duchenne muscular dystrophy (DMD) is a monogenic and fatal genetic disorder characterized by muscle wasting, loss of ambulation, and death by the age of 20 due to the loss of functional dystrophin - an essential musculoskeletal protein. Gene therapy has held promise for treating monogenetic diseases, however, delivery barriers and the requirements for costly repetitive treatments are a few of the challenges of technologies in the therapeutic pipeline. Recently, the CRISPR/Cas9 system has been adapted for genome editing to make specific modifications in the host genome. This technology would enable permanent genome corrections and a potentially curative approach to DMD.
Previously we adapted CRISPR/Cas9 for targeted deletions of regions of the human dystrophin gene that restore expression of a functional protein (Ousterout et al., Nature Communications (in press)). The delivery of Cas9 with single guide RNAs (sgRNAs) targeting the introns surrounding exons 51 and 45-55 created double strand breaks which are repaired through non-homologous end joining to create genomic deletions of these regions. These deletions restored the reading frame and recovered dystrophin expression in cultured patient-derived myoblasts. Therapeutically, this approach could be used to convert the fatal DMD phenotype to the milder phenotype associated with partial dystrophin function characteristic of Becker muscular dystrophy.
Here we utilize adeno-associated virus (AAV) to deliver the CRISPR/Cas9 system to the mdx mouse model of DMD. AAV vectors were designed to package and deliver genes encoding the Staphylococcus aureus Cas9 (SaCas9), due to its smaller size (3.2 kb) compared to the widely used Cas9 from Streptococcus pyogenes. Two single guide RNAs (sgRNAs) were designed to target sites flanking exon 23 of the dystrophin gene and validated to direct SaCas9-mediated deletion of this exon, which contains a premature stop codon in this model. Mice were anesthetized and AAV1 containing the SaCas9 and sgRNA expression cassettes was injected into the gastrocnemius muscle of 8 week old mice. At 4 and 8 weeks post-injection, the gastrocnemius was harvested and genomic DNA and mRNA were extracted. PCR of the genomic DNA demonstrated the expected ∼1200 bp deletion of exon 23. Further, RT-PCR of the extracted mRNA showed removal of the 216 bp encoding exon 23. Furthermore, sgRNAs compatible with SaCas9 have been developed and validated for the deletion of human exon 51.
This work demonstrates proof-of-principle of CRISPR/Cas9-mediated genome editing in skeletal muscle in an adult mammal, opening up new possibilities for gene therapy and the study of gene function. Optimization of delivery and activity is underway to improve the therapeutic potential of this approach. Additional work to characterize functional improvement in the mouse hind limb is also ongoing. This work establishes CRISPR/Cas9-mediated genome editing as a potential therapeutic approach to DMD.

14) Sustained Expression of a Canine Micro-Dystrophin Lead To Improvement of Limb Muscle Function in Dystrophic Dogs Following Large Scale AAV-Mediated Treatment

Christine Halbert, Melissa Goddard, Donghoon Lee, David Mack, Tiffany Butts, Robert Grange, Mackenzie Johnson, Rainer Storb, Dusty Miller, Jeffrey Chamberlain, Stephen Tapscott, Martin Childers, Zejing Wang. Fred Hutchinson Cancer Research Center, Seattle, WA; University of Washington, Seattle, WA; Virginia Tech, Blacksburg, VA

Adeno-associated viral (AAV) vectors as gene delivery vehicles have shown promise both in preclinical studies and clinical trials for a number of acquired and inherited diseases, including Duchenne Muscular Dystrophy (DMD). Studies have shown that host immune responses against AAV can compromise vector delivery and prevent sustained therapeutic gene expression in animal models and humans. We developed and demonstrated a novel AAV production system that prevents aberrant packaging of the AAV capsid (cap) genes. Compared to conventional AAV production methods, our new approach eliminates unwanted in vivo expression of AAV capsid proteins and significantly reduces host immune responses. Here, we demonstrated in a pilot study that we were able to reduce a triple-drug immunosuppressive regimen to a mild 2-drug regimen while retaining sustained canine micro-dystrophin expression in CXMD dogs following large scale delivery of an AAV vector carrying the canine micro-dystrophin to a group of muscles in the hind limb. Amelioration of muscle pathology as a result of sustained expression of the therapeutic gene was demonstrated by histological analysis of muscle tissues and by non-invasive magnetic resonance imaging (MRI). Further, we determined functional benefit of micro-dystrophin expression by muscle force measurement and gait analysis. Significant improvement in muscle force, as indicated by more than 100% increase in strength (torque), was observed in the treated limb compared to that in the untreated limb. In conclusion, by combining newly improved AAV production and mild clinically applicable immunosuppression, the finding of long term robust dystrophin expression in CXMD dogs' limb with significant functional improvement opens the possibility of translating these strategies to a human DMD trial, which can improve patients' immediate quality of life.

15)  Improved Transduction of Canine X-Linked Muscular Dystrophy With rAAV9-Microdystrophin By Using MSCs Pretreatment

Hiromi Hayashita-Kinoh, Hironori Okada, Yuko Nitahara-Kasahara, Tomoko Chiyo, Kiwamu Imagawa, Katsuhiko Tachibana, Shin'ichi Takeda, Takashi Okada. Dept. of Molecular Therapy, National Institute of Neuroscience, NCNP, Tokyo, Japan; Department of Biochemistry and Molecular Biology, Nippon Medical School, Tokyo, Japan; JCR Pharmaceuticals Co., Ltd., Hyogo, Japan

Background: Duchenne muscular dystrophy (DMD) is a congenital disease causing progressive deterioration of skeletal and cardiac muscles because of mutations in the dystrophin gene. Supplementation of dystrophin using rAAV is effective to improve pathogenesis of animal models of DMD. However, we have previously reported that local injection of rAAV2 or rAAV8 to canine skeletal muscles without immunosuppression resulted in insufficient transgene expression with potent immune responses. Mesenchymal stem cells (MSCs) regulate graft-versus-host disease (GVHD) by virtue of their immunosuppressive effects. Short-term exposure of MSCs with vector administration efficiently inhibited immune responses to the rAAV capsid and/or transgene products. 
Methods: Human bone-marrow derived MSCs and rAAV9-Luciferase were intravenously injected into the normal dog at 8 weeks old. Seven days after injection, hMSCs were systemically injected again. At 8 days after 1st injection, rAAV9-Luciferase was intramuscularly injected into the tibialis anterior muscle of the same dog. To examine the immune response against rAAV, purified canine peripheral leukocytes were exposed to rAAV9 for 4 hours, and then IFN-γ expression was analyzed using qRT-PCR. Skeletal muscles of the rAAV-Luc injected animals were sampled by biopsy for expression analysis at 4 weeks after intramuscular injection. 
Results: Intramuscular injection of rAAV-Luc following hMSCs treatment, resulted in higher expression of Luciferase at the injected muscle, compared to the rAAV-Luc transduction alone. Expression of IFN-γ in the purified peripheral blood leukocytes after the rAAV exposure were not induced in the rAAV-Luc with MSCs, suggesting the immune suppressive effects of the MSCs.
Conclusion: Our results demonstrate that rAAV-Luc injection with MSCs treatment improved expression of rAAV-derived Luciferase in dogs. This strategy would be effective approach to analyze the expression and function of transgene in vivo. These findings also support the future feasibilities of rAAV-mediated protein supplementation strategies.

16) Mesenchymal Stromal Cells Can Ameliorate the Progressive Phenotype of Dog With Duchenne Muscular Dystrophy

Yuko N. Kasahara, Hiromi H. Kinoh, Mutsuki Kuraoka, Tomoko Chiyo, Hironori Okada, Nana Tsumita, Kiwamu Imagawa, Katsuhiko Tachibana, Shin'ichi Takeda, Takashi Okada. Molecular Therapy, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Kodaira, Tokyo, Japan; Biochemistry and Molecular Biology, Nippon Medical School, Bunkyo-ku, Tokyo, Japan; JCR Pharmaceuticals Co., Ltd., Ashiya, Kobe, Japan

Background: Duchenne muscular dystrophy (DMD) is an incurable genetic disease with early mortality that exhibits skeletal muscle weakness with chronic inflammation. Multipotent mesenchymal stromal cells (MSCs) could be a potential therapeutics because of their immunosuppressive properties and ability of differentiation to myogenic cells in situ. In the present study, we examined the strategies for effective cell transplantation to develop a novel approach for functional recovery of the skeletal muscles using a dog model of Duchenne muscular dystrophy. 
Methods: Human bone marrow-derived MSCs were intravenously injected into two littermates of canine X-linked muscular dystrophy in Japan (CXMDJ) at weekly interval for 8 weeks without immunosuppression. Clinical phenotypes on transplanted dogs were analyzed by using blood exams, physical capacity, MRI and histology compared with two non-injected littermates as controls. 
Results: The downregulation of inflammation in the lower legs of MSCs-treated CXMDJ was confirmed by magnetic resonance imaging analysis. Immunofluorescence analysis showed decreased area of IgG-positive necrotic myofibers and also increased number of developmental myosin heavy chain-positive regenerative fibers. Impaired tetanic force of gastrocnemius of CXMDJ recovered in hMSCs-treated CXMDJ, from 25% to 80% of wild type. Although CXMDJ showed progressive muscle atrophy in the all four limbs, exercise intolerance and abnormal locomortion, we observed improved phenotypes in the hMSCs-treated CXMDJ along with the improved pace of flip-flop and running. 
Conclusion: We demonstrated that the systemic injection of MSCs ameliorates the progressive phenotype in CXMDJ. The therapeutic effects might be associated with the production of paracrine or endocrine factors that regulate inflammation, and might also stimulate the proliferation of endogenous stem cells at the injured muscle tissue. This strategy of MSCs treatment would be promising for the future DMD cell therapy.

17) Development of Human Artificial Chromosomes for Gene Cell Therapy of Muscular Dystrophies

Artur A. Isaev, Ilya I. Eremin, Andrey A. Pulin, Roman V. Deev, Alexey N. Tomilin, Michail A. Liskovykh, Vladimir Larionov, Natalia Kouprina, Kseniya Iur'eva, Vadim L. Zorin, Ilya Ya Bozo, Konstantin V. Kotenko. Human Stem Cells Institute OJSC, Moscow, Russian Federation; State Research Center - Burnasyan Federal Medical Biophysical Center FMBA of Russia, Moscow, Russian Federation; Institute of Cytology of Russian Academy of Sciences, Saint Petersburg, Russian Federation; National Cancer Institute NIH, Bethesda

Muscular dystrophies are clinically heterogeneous group of muscle diseases related to different genes defects. The results of defects impair myogenic differentiation and histophysiology of skeletal muscles. One of major forms of disease - limb-girdle muscular dystrophy 2B (LGMD2B), caused by dysferlin gene mutations. Both big size of gene and wide variety of mutations complicate development of genetically engineered drugs. Human artificial chromosomes (HAC) could be used as alternative approach. This vector can contain unlimitedly large DNA region for transfer, is retained during cell division, and does not have carcinogenic and immunogenic properties. 
HAC matrix was developed earlier by Larionov V. et al. ( Optimized DYSF gene sequence (optDYSF) from pAd/CMV/V5-DEST vector (kindly provided by A.A. Rizvanov) was transferred into backbone vector also containing loxP sites for further transfer into HAC. Transfer correctness confirmed by sequencing. After that Chinese hamster ovary cells carrying HAC were transfected by backbone vector containing optDYSF. Selection performed in HAT medium. 7 clones were selected and analyzed at day 10. Expression of optDYSF observed in all obtained clones. Episomal HAC localization determined using fluorescent hybridization in sity by colocalization of label at optDYSF and a-satellite DNA. Episomal localization of HAC determined only in 2 clones from 7. Dysferlin production observed in all clones.
On other hand described vector requires somatic myogenic vector cell. Human myoblasts could be successfully used for this purpose. The major source of myoblasts - skeletal muscle tissue is not optimal due to limited proliferative potential, especially in patients with muscular dystrophies. Our team revealed presence of myoblasts in gingival mucosa specimens and developed new method of myoblasts obtainment from it. Primary cultures of multipotent mesenchymal stromal cells (MSC) were derived from gingiva biopsy samples. The cells expressed typical mesenchymal stem cells markers and had ability for 3-way differentiation. Myogenic induction of gingiva derived MSC in culture was demonstrated. Gingiva derived MSC are capable to express markers of myogenic differentiation (skeletal actin, sceletal myosin, MyoD1) and form multicellular elongated fibers - myotubes. Ability of gingiva derived MSC cultures to differentiate in myogenic direction was preserved during up to 10th passage. Such features make this cell source attractive for using in treatment of inherited muscular degenerative diseases.
In our future research we are planning to transfer HAC containing optDYSF gene into clonogenic gingiva derived MSC using microcell-mediated chromosome transfer. This method could be used for further development of gene cell therapy of LGMD2B.

18)  The MDX/UTR +/- Mouse Is a Superior Mouse Model for Duchenne Muscular Dystrophy

Thais B. Lessa, Chady H. Hakim, Carlos E. Ambrósio, Dongsheng Duan. Department of Surgery, University of São Paulo, São Paulo, Brazil; Department of Molecular Microbiology and Immunology, University of Missouri, Columbia; Department of Veterinary Medicine, University of São Paulo, Pirassununga, Brazil

Duchenne Muscular Dystrophy (DMD) is a severe muscle wasting disease caused by the absence of the dystrophin protein. Dystrophin, the core component of the dystrophin-associated glycoprotein complex, is responsible in stabilizing the sarcolemma during muscle contraction and relaxation. In the absence of dystrophin, muscle becomes fragile and susceptible to damage. This results in progressive muscle degeneration and replacement with fibrotic tissue. The mdx mouse is the most commonly used animal model for DMD. However, it is also considered a poor model because it cannot reproduce the severe dystrophic phenotype seen in patients. Genetic elimination of utrophin, an autosomal homolog of dystrophin, yields a symptomatic double knockout (dko) model. Unfortunately, human patients do not have utrophin deficiency. An utrophin heterozygous mdx (mdx/utrn+/-) mouse would theoretically be a better model should it show more severe muscle dysfunction than the mdx mouse. To test this hypothesis, we systematically examined diaphragm disease in 2-m-old sex-matched mdx and mdx/utrn+/-mice. Compared to the mdx mouse, the mdx/utrn+/-mouse displayed more defective histological changes. Importantly, muscle force and passive property were significantly more compromised in the mdx/utrn+/-mouse. Collectively, our data demonstrated for the first time that the mdx/utrn+/-mouse represent a better DMD model at both morphological and physiological levels.

19) Treatment of DMD 5' Mutations Through Two Different exon2 Skipping Strategies: Intramuscular Delivery of rAAV9.snRNA Mediated Skipping and Antisense Morpholino Oligomers

Tabatha R. Simmons, Nicolas Wein, Adeline Vulin-Chaffiol, Kristin Heller, Andrea Rutherford, Louise Rodino-Klapac, Kevin M. Flanigan. Center for Gene Therapy, The Research Institute at Nationwide Childrens, Columbus, OH; Molecular, Cellular, Developmental Biology, The Ohio State University, Columbus, OH; Pediatrics, The Ohio State University, Columbus, OH; Neurology, The Ohio State Univeristy, Columbus, OH

To date, exon-skipping therapies for Duchenne muscular dystrophy (DMD) patients have focused on patients with out-of-frame exon deletions, in whom treatment results in larger but in-frame internal deletions which lead to translation of an internally truncated but partially functional dystrophin protein. We are developing exon-skipping therapies for exon duplication mutations, accounting for around 6% of all mutations, with the intent to induce production of wild-type transcripts and protein. As a model of the most common single exon duplication, we have developed a new mouse with a duplication of exon 2 (the Dup2 mouse) that largely recapitulates the findings in the standard mdx mouse. Using this mouse, we are testing both virally (AAV) mediated skipping induced by a modified U7snRNA (rAAV9.U7.ACCA), and antisense oligomer-induced skipping. Intramuscular injections of the tibialias anterior (TA) (N=6 muscles each) were performed rAAV9.U7.ACCA at 6 doses between 1x1010 and 1x1012 total vector genomes. Intramuscular (TA) injections of an exon 2-directed antisense peptide-morpholino conjugate (PPMO) were performed at doses of either 10 or 20 ug total PPMO (N=3 each). For each study, mice were sacrificed 1 month post injection and muscle analysis of DMD mRNA and dystrophin protein expression. Treatment with either modality results in significant skipping of the duplicated exon 2, and expression of a functional dystrophin isoform at levels of up to 30% of normal with AAV-mediated skipping. Physiology has been assessed in the AAV-treated mice, in which correction of TA absolute force deficits in comparison to the background Bl6 strain are seen, along with a partial yet significant correction of eccentric contraction injury in comparison to untreated Dup2 mice. These data suggest that skipping of a duplicated exon 2 may be a feasible therapeutic approach, particularly because skipping of exon 2 may be associated with an apparently unlimited therapeutic window. Over-skipping – to the exclusion of exon 2 entirely – results in activation of an internal ribosome entry site (IRES) located in exon 5 of dystrophin that allows for cap-independent translation from an alternative initiation site within exon 6. This alternate dystrophin isoform is highly functional despite being N-truncated, consistent with the observation that patients expressing it have minimally symptomatic (or even asymptomatic) Becker muscular dystrophy (BMD), and suggesting a potential route to therapy for any of the approximately 5% of patients with mutations in the 5' end of the gene.

20) β-Sarcoglycan Gene Transfer Prevents Muscle Fibrosis and Inflammation in an Aged LGMD2E Mouse Model

Eric R. Pozsgai, Danielle A. Griffin, Kristin N. Heller, Jerry R. Mendell, Louise R. Rodino-Klapac. Center for Gene Therapy, Nationwide Childrens Hospital, Columbus, OH; Pediatrics, The Ohio State University College of Medicine, Columbus, OH; Biomedical Sciences Graduate Program, The Ohio State University College of Medicine, Columbus, OH

Limb-girdle muscular dystrophy type 2E (LGMD2E) results from mutations in the β-sarcoglycan (SGCB) gene causing loss of a structural protein component of the dystrophin-associated protein complex (DAPC) located at the sarcolemma. This loss results in histopathological features including chronic muscle fiber necrosis, inflammation, fat replacement and fibrosis, accompanied by deteriorating muscle strength and function. To date, no effective therapy exists to treat this debilitating disease. A suitable model for translational studies is the murine model of LGMD2E that completely lacks β-sarcoglycan (Sgcb-null mouse), and has clinical-pathological features in skeletal and cardiac muscle that replicate the human disease. Considering that a major question unanswered by gene replacement therapy is the potential efficacy of gene replacement once significant degrees of connective tissue have infiltrated dystrophic muscle, the studies described here in the β-sarcoglycan knock out mouse have particular relevance for planning future clinical trials. On the clinical side, fibrosis presents a major impasse for functional recovery. In this study, we directly addressed this question using a codon optimized human β-sarcoglycan gene (hSGCB) driven by a muscle specific tMCK promoter and the AAVrh.74 serotype (scAAVrh74.tMCK.hSGCB). We first showed restoration of expression 12 weeks post treatment following a direct intramuscular injection which was accompanied by improvements in histological parameters. Furthermore, following a clinically relevant isolated limb vascular delivery (5 X10^11 vg) we found that >90% of muscle fibers expressed β-sarcoglycan in lower limb muscles. Histopathology showed a decrease in central nucleation and normalization of muscle fiber size. Immunohistochemical staining for various immune components including CD3, CD4, CD8, and macrophages showed a reduction in the numbers of immune cells following treatment. The restoration of β-sarcoglycan expression and improvement in overall histology also correlated with improvement in functional outcomes assessed by absolute and specific force generation and resistance to contraction induced injury. To measure fibrosis, quantification of picrosirius stained sections revealed a reduction of collagen deposition in TA (from 40.72 ± 1.40% to 21.22 ± 1.09%, p<0.01) and gastrocnemius (37.92 ± 1.25% to 22.90 ± 0.99%, p<0.01). Moreover, we treated an additional cohort of aged 6 month old Sgcb-null mice with extensive fibrosis already present. At 9 months >80% of muscle fibers were transduced which was accompanied by a significant reduction of fibrosis similar to what was achieved following vascular delivery in younger mice. This pre-clinical study addresses the potential for gene replacement to reverse the debilitating fibrosis, typical of many of the muscular dystrophies providing momentum for movement to a clinical gene replacement for LGMD2E.

21) Alpha-Dystrobrevin-3 Prevents Myopathy and Restores Diaphragm Function in the Alpha-Dystrobrevin Null Mouse

Guy L. Odom, Glen B. Banks, Marvin E. Adams, Kenneth L. Bible, Stanley C. Froehner, Jeffrey S. Chamberlain. Neurology, University of Washington, Seattle, WA; Physiology and Biophysics, University of Washington, Seattle, WA

The dystrophin-glycoprotein complex (DGC) primarily functions as a structural element enabling a linkage between the cytoskeleton and the extracellular matrix. Alpha-dystrobrevin (α-Db), a dystrophin subfamily member, is a component of the DGC within striated muscles. Decreasing amounts of α-Db from the sarcolemma contributes to severity of disease in several muscular dystrophies including Duchenne muscular dystrophy (DMD). The functional roles of α-Db1 and α-Db2 in muscle have been well studied. However, the role of α-Db3 has largely been ignored because it lacks many of the known functional domains present in α-Db1 and α-Db2. Importantly, we demonstrate by whole-body adeno-associated viral vector gene transfer into α-dystrobrevin-null mice, that α-DB3 becomes integrated within the DGC, localizes to costameres, prevents muscle degeneration, synapse fragmentation, myotendinous junction defects and corrects diaphragm functional capacity. Further, we demonstrate α-Db3 can bind the sarcoglycan complex in vitro. Thus, the association of α-Db3 within the DGC appears essential for its function, providing a direct link similar to dystrophin, with transmembrane and cytoplasmic DGC components whose absence likely contributes to sarcolemma fragility and/or disruption of signaling pathways in multiple muscular dystrophies.

22) Characterization of IPSC-Derived Myogenic Progenitors Isolated from Mouse Models of Duchenne Muscular Dystrophy

Niclas E. Bengtsson, John K. Hall, André M. Lieber, Jeffrey S. Chamberlain. Department of Neurology, University of Washington, Seattle, WA; Division of Medical Genetics, University of Washington, Seattle, WA

Duchenne muscular dystrophy (DMD) affects approximately 1 in 5000 children and is characterized by progressive muscle wasting, weakness and premature death. DMD is caused by mutations in the gene encoding dystrophin, which plays an essential role in maintaining muscle integrity by providing a structural link between the cytoskeleton and the extracellular matrix. In the absence of dystrophin, affected muscles experience repeated bouts of injury, inflammation and necrosis, resulting in reduced regenerative capacity and the replacement of muscle mass with fibrotic and adipose tissue. Both gene and cell therapies are candidate approaches to replace the defective dystrophin gene. While gene therapy has the potential to halt disease progression in targeted muscles, cell therapies could both introduce replacement genes and generate new muscle or muscle stem cells to affected tissues. Cell therapies for DMD have thus far shown moderate success due to limited ex vivo expansion potential of progenitor cells, poor survival and migration of cells following transplantation, inefficient myogenic conversion of non-myogenic cells as well as host rejection of donor cells. Most of these limitations could potentially be addressed using gene corrected induced pluripotent stem (iPS) cells derived from biopsies of affected patients.
We have established and characterized several iPSC lines from control, dystrophic (mdx) and transgenic-mdx mice. Selected iPS cells expressed markers associated with pluripotency, exhibited normal karyotypes and were able to form all three germ layers during in vivo teratoma assays. To generate myogenic progenitors from iPS cells, we have investigated the use of an inducible form of the myogenic regulatory factor MyoD (MyoD-ER[T]). Upon transient activation of MyoD-ER[T], iPSCs are readily converted into myogenic progenitor cells that can efficiently differentiate into myocytes and fuse into myotubes in vitro. iPS cells derived from dystrophic mice and which carry different versions of dystrophin expression cassettes can engraft into mdx muscles in vivo and generate dystrophin-positive myofibers. We are currently comparing the efficiency of various myogenic cell populations resulting from MyoD-ER[T] induction to generate functional myogenic engraftment in vivo following transplantation into mdx mouse hosts. We are also comparing the relative efficiency of dystrophin production in myogenic cells derived from mdx iPSCs using gene replacement vs. gene modification approaches. Together, these approaches show potential for ex vivo gene therapy of DMD using dystrophin-corrected autologous cell transplantation.

23) Histologic Assessment of Geriatric Esophagus Revels Therapeutic Targets in Wild-Type and Dystrophic Mouse Models

John K. Hall, Ladan L. Mozaffarian, Jeffrey S. Chamberlain. Neurology, University of Washington, Seattle, WA

Gastroesophageal disorders increase as a process of aging and are exacerbated in degenerative neuromuscular disease such as Duchenne muscular dystrophy (DMD). DMD affects ∼1/5000 male births and is a catastrophic and ultimately fatal muscle wasting disease resulting from a mutation in the gene encoding the integral muscle protein dystrophin. Advancements in the treatment for DMD patients has increased patient lifespan and underscores a need to assess the impact of the disease in a range of organ systems, including the esophagus, likely to present new therapeutic challenges over time. Severity of esophageal pathology is varied to include reflux disease, achalasia, and megaesophagus. Defined as a pathologic enlargement of the esophagus, megaesophagus can result in a failure to complete peristalsis, impaction, severe weight loss and increased morbidity. Although poorly defined in human DMD patients, megaesophagus has been documented in canine and mouse models of DMD, however, a detailed cellular and mechanistic understanding remains unclear. We have performed a comprehensive morphologic and histologic examination of wild-type (C57/BL6) and dystrophic (mdx4cv) mouse esophagus to determine onset, pathology and changes in cellular composition accompanying megaesophagus onset. Age-associated presentation of megaesophagus was highly penetrant in both mouse models, with a four-fold increase in lumen dilation and coincident rate of impaction seen in aged dystrophic mice. In comparison, skeletal muscle area increased in both models with age, however, wild-type mice presented significantly greater hypertrophy than that observed in dystrophic mice. Interestingly, strain specific differences were not identified in esophagus mucosa strata with both strains presenting significant and similar increases in mucosa area and aging-dependent changes in mucosa composition. Further, a pronounced, aging-specific degradation of smooth musculature was observed in both mouse strains independent of dystrophin expression within wild-type mice. We demonstrate that esophageal smooth muscle is targetable via AAV-mediated gene therapy with future efforts aimed at correcting degeneration present within geriatric wild-type and dystrophic mouse models.

24) Efficient Reconstitution of a 7kb Therapeutic Mini-Dystrophin Gene in Duchenne Muscular Dystrophy Dog Muscle

Kasun Kodippili, Xiufang Pan, Hsiao Yang, Chady Hakim, Yadong Zhang, Yongping Yue, Dongsheng Duan. Molecular Microbiology and Immunology, University of Missouri - Columbia, Columbia, MO; Biomedical Sciences, University of Missouri - Columbia, Columbia, MO

Duchenne muscular dystrophy (DMD) is a devastating muscle wasting disease caused by mutations in the dystrophin gene. Restoring dystrophin expression by adeno-associated virus mediated gene therapy holds great promise as a mutation-independent therapy for all DMD patients. The full-length dystrophin cDNA (∼11 kb) is far beyond the 5 kb packaging capacity of a single AAV vector. Consequently, truncated micro- and mini-dystrophin genes have been used. The micro- and mini-dystrophin genes encode ∼30% and ∼50% of the full-length dystrophin coding sequence, respectively. Mini-dystrophin is expected to provide better protection than micro-dystrophin. We have previously shown that dual AAV vectors can efficiently express a 6 to 8-kb mini-dystrophin in mdx mice. Dual AAV-mediated mini-dystrophin gene therapy significantly ameliorated histopathology and improved muscle function in mdx mice, a mouse model of DMD. To translate this promising therapy to large mammals, here we evaluated the reconstitution of a 7-kb minigene in the canine model of DMD by local injection. We engineered a pair of dual-AAV vectors to express a 7-kb canine codon-optimized ΔH2-R15 mini-dystrophin gene. For easy detection, a flag-tag and a GFP gene were fused to the N-terminal and C-terminal ends, respectively. To determine whether dual AAV vectors can lead to efficient mini-dystrophin expression, we co-delivered both vectors to the extensor carpi ulnaris muscle in the forelimb of DMD dogs at a dose of 2×1013 vg particles/vector/muscle. Two months after gene transfer we evaluated transduction efficiency and observed successful expression of mini-dystrophin from the dual-AAV vectors. All flag-tag positive myofibers were also positive for minidystrophin, GFP and dystrophin-associated glycoprotein complex proteins. Importantly, mini-dystrophin gene therapy also reduced muscle force loss under the stress of repeated cycles of eccentric contraction. These results establish the proof-of-concept for mini-dystrophin gene therapy in dystrophic muscles of large mammals.

25)  Efficient Reconstitution of a 7kb Therapeutic Mini-Dystrophin Gene in Duchenne Muscular Dystrophy Dog Muscle

Kasun Kodippili, Xiufang Pan, Hsiao Yang, Chady Hakim, Yadong Zhang, Yongping Yue, Dongsheng Duan. Molecular Microbiology and Immunology, University of Missouri - Columbia, Columbia, MO; Biomedical Sciences, University of Missouri - Columbia, Columbia, MO

Duchenne muscular dystrophy (DMD) is a devastating muscle wasting disease caused by mutations in the dystrophin gene. Restoring dystrophin expression by adeno-associated virus mediated gene therapy holds great promise as a mutation-independent therapy for all DMD patients. The full-length dystrophin cDNA (∼11 kb) is far beyond the 5 kb packaging capacity of a single AAV vector. Consequently, truncated micro- and mini-dystrophin genes have been used. The micro- and mini-dystrophin genes encode ∼30% and ∼50% of the full-length dystrophin coding sequence, respectively. Mini-dystrophin is expected to provide better protection than micro-dystrophin. We have previously shown that dual AAV vectors can efficiently express a 6 to 8-kb mini-dystrophin in mdx mice. Dual AAV-mediated mini-dystrophin gene therapy significantly ameliorated histopathology and improved muscle function in mdx mice, a mouse model of DMD. To translate this promising therapy to large mammals, here we evaluated the reconstitution of a 7-kb minigene in the canine model of DMD by local injection. We engineered a pair of dual-AAV vectors to express a 7-kb canine codon-optimized ΔH2-R15 mini-dystrophin gene. For easy detection, a flag-tag and a GFP gene were fused to the N-terminal and C-terminal ends, respectively. To determine whether dual AAV vectors can lead to efficient mini-dystrophin expression, we co-delivered both vectors to the extensor carpi ulnaris muscle in the forelimb of DMD dogs at a dose of 2×1013 vg particles/vector/muscle. Two months after gene transfer we evaluated transduction efficiency and observed successful expression of mini-dystrophin from the dual-AAV vectors. All flag-tag positive myofibers were also positive for minidystrophin, GFP and dystrophin-associated glycoprotein complex proteins. Importantly, mini-dystrophin gene therapy also reduced muscle force loss under the stress of repeated cycles of eccentric contraction. These results establish the proof-of-concept for mini-dystrophin gene therapy in dystrophic muscles of large mammals.

26)  IntraCSF Administration of AAV9/MeCP2 Extends Lifespan of MeCP2-Null Mice While Preventing Toxicity Associated With IV Administration

Sarah E. Sinnett, Sahana N. Kalburgi, Steven J. Gray. Gene Therapy Center, University of North Carolina at Chapel Hill, Chapel Hill, NC; Biomedical Research Education and Training, Vanderbilt University, Nashville, TN

Rett Syndrome (RTT) is a neurodevelopmental disorder that shortens the lifespan of patients while depriving them of the ability to walk, talk, or interact with others. RTT is caused by inactivating mutations in the X chromosome-linked gene encoding methyl-CpG-binding protein 2 (MeCP2), a transcription regulator that is highly expressed in neurons. Patients with RTT express mutant MeCP2 in ∼50% of their cells due to random X-chromosome inactivation in each cell. The remaining “healthy” cells have an activated chromosome encoding functional (WT) MeCP2. Although researchers have already used MeCP2 gene therapy to reverse the RTT phenotype in mosaic mice, concerns about side effects (i.e., motor and learning deficits) resulting from MeCP2 overexpression in “healthy” cells persist. Indeed, humans suffering from MeCP2 duplication syndrome have been shown to suffer from muscular spasticity and intellectual disabilities. In addition to persistent concerns about MeCP2 overexpression, IV administration of the scAAV9/hMeCP2 vector (1x1011 vg/mouse) has been shown to cause significant liver toxicity in MeCP2-null mice (Gadalla et al, Mol Ther, 2013). We tested the hypothesis that by moving from an IV route of administration to an intraCSF route, we could lower the dose of AAV vector enough to avoid the high peripheral organ transduction that generates toxic side effects. Modeling our earlier experiment with an IV route, juvenile (4-5 week old) knock-out (KO) mice and WT littermates received a single intrathecal injection of AAV9/MeCP2 vector. RTT mice dosed intrathecally with 1x1010 vg had a 48% increase in median survival compared to vehicle-injected mice (n=6 treated; n=11 vehicle; P=0.008), nearly identical to the rescue we previously observed following the IV approach but at 1/10th the dose and avoiding overexpression-related toxicity in the liver. In conclusion, by changing the route of administration of the AAV9/MeCP2 vector from IV to intraCSF, we were able to generate a nearly identical therapeutic outcome with a 10-fold lower dose, and without excessive (toxic) gene transfer to peripheral organs. As a next step to utilize the intraCSF route of administration, DNA shuffling and directed evolution were utilized to select for AAV capsid variants that would have higher efficiency and improved biodistribution in the RTT mice after intraCSF administration. Multiple clones were selected that have CNS transduction equivalent to AAV9, but importantly, all had highly reduced peripheral organ biodistribution. Preliminary studies using two clones, RTTF1.04 and RTTF1.11, were able to confer significant survival benefits to male RTT KO mice (34% and 25% prolongation of lifespan, P = 0.03 and 0.05, respectively) after delivery of the MeCP2 transgene at a dose of 1x1010 vg. Overall, our studies support the use of an intraCSF route of administration to treat RTT using AAV vectors, and the potential to develop safer and more effective AAV-based vectors tailored to the intraCSF route of administration. 

27) Systemic Delivery of Dysferlin Overlap Vectors Mediates Functional Recovery of Dysferlin Deficiency

Patricia C. Sondergaard, Danielle A. Griffin, Eric R. Pozsgai, Ryan Johnson, Kim Shontz, Jerry R. Mendell, Louise R. Rodino-Klapac. Center for Gene Therapy, Nationwide Children's Hospital, Columbus, OH; Biomedical Sciences Graduate Program, The Ohio State University, Columbus, OH; Department of Pediatrics, The Ohio State University, Columbus, OH; Department of Neurology, The Ohio State University, Columbus, OH

Dysferlinopathies comprise a family of disorders caused by mutations in the dysferlin (DYSF) gene leading to absent or mutant protein. Dysferlin protein has been implicated in multiple functional roles specifically in membrane stabilization/repair, t-tubule formation and vesicle trafficking. The loss of dysferlin causes a progressive dystrophy characterized by chronic muscle fiber loss, fat replacements and fibrosis resulting in deteriorating muscle weakness. Efforts made in the gene therapy arena with dysferlin or surrogate gene replacement have shown some efficacy in restoring membrane repair, however, only delivery of full-length DYSF has been able to correct the underlying histopathology. Therefore, there is a strong rationale to develop therapies that deliver the entire DYSF cDNA. A potential issue with this is the size of the DYSF gene (6.5 kb) which is too large for canonical AAV packaging. To circumvent this, we have developed and previously shown efficacy with a unique dual vector system using AAV to deliver and express DYSF specifically in muscle cells. This two vector system (AAV.DYSF.DV) packaged in the rh.74 serotype is defined by a 1 kb region of homology between the two vectors. Following delivery to muscle, this overlap serves as a substrate for recombination/repair to generate the full-length gene. Our previous work studied the efficacy of this treatment strategy through intramuscular and regional vascular delivery routes. However, as generalized muscle weakness is common in dysferlinopathies, therapies targeting all muscle groups are warranted to maximize clinical efficacy. In this update, we have treated dysferlin-deficient mice systemically by intravenous injection to target all muscles through the vasculature for efficacy and safety studies. Mice were evaluated at 3 and 6 months post-treatment for dysferlin expression, restoration of membrane repair capability, diaphragm specific force measurements and muscle histology. Additional animals are awaiting MRI analysis following 1 year of treatment. A single systemic dose of 6x1012 vector genomes (3x1012 vg of each vector) resulted in widespread gene expression exceeding 30% of muscle fibers. Treated muscles showed a significant decrease in central nucleation and collagen deposition. Membrane repair ability was improved toward wild-type and force deficits in the diaphragm were restored to wild-type levels. The mice showed no evidence of local or systemic toxicity, further confirming previous safety data. This study, in conjunction with our previous work, lays the foundation for clinical trial.

28) A Comparison of AAV Strategies Distinguishes Overlapping Vectors for Efficient Systemic Delivery of the 6.2kb Dysferlin Coding Sequence

William Lostal, Marina Pryadkina, Isabelle Richard. R&D, Genethon, Evry, France

Recombinant AAV (rAAV) is currently the best vector for gene delivery into the skeletal muscle. However, the 5-kb packaging size of this virus is a major obstacle for large gene transfer. This past decade, many different strategies were developed to circumvent this issue (concatemerization-splicing, overlapping vectors, hybrid dual or fragmented AAV). Loss of function mutations in the DYSF gene whose coding sequence is 6.2kb lead to progressive muscular dystrophies (LGMD2B: OMIM_253601; MM: OMIM_254130; DMAT: OMIM_606768). In this study, we compared large gene transfer techniques to deliver the DYSF gene into the skeletal muscle. After rAAV8s intramuscular injection into dysferlin deficient mice, we showed that the overlap strategy is the most effective approach to reconstitute a full-length messenger. After systemic administration, the level of dysferlin obtained on different muscles corresponded to 0.5 to 2 fold compared to the normal level. We further demonstrated that the overlapping vector set was efficient to correct the histopathology, resistance to eccentric contractions and whole body force in the dysferlin deficient mice up to one year after injection. Altogether, these data indicate that using overlapping vectors could be a promising approach for a potential clinical treatment of dysferlinopathies.