ABSTRACTS THAT WILL BE PRESENTED IN THE 9TH ANNUAL MEETING OF THE AMERICAN SOCIETY OF GENE THERAPY, 2006, MAY 31- JUNE 4, BALTIMORE

1) Systemic Administration of rAAV6-Microdystrophin Preserves Muscle Function and Extends Lifespan in the Dystrophin-/Utrophin- Mouse Model of Severe Muscular Dystrophy

Paul Gregorevic, Michael J. Blankinship, Elina Minami, James M. Allen, Charles E. Murry, Jeffrey S. Chamberlain Department of Neurology, University of Washington, Seattle, WA; Department of Pathology, University of Washington, Seattle, WA

Gene replacement is a promising strategy for treating severe neuromuscular disorders such as Duchenne muscular dystrophy (DMD). However, the therapeutic potential of miniaturized dystrophin expression cassettes has not been rigorously assessed because traditional gene transfer systems cannot simultaneously transduce the important skeletal muscles, and the heart of animals that model muscular dystrophies. Furthermore, because of limited prospects for efficient gene transfer, previous studies have typically assessed the effects of localized interventions in the comparatively robust dystrophin-deficient mdx mouse, which poorly models the progressive, severe dystrophy that shortens the lives of DMD patients. We recently established a method that facilitates the delivery of genes to striated muscles throughout adult animals via intravenous administration of recombinant adeno-associated viral vectors (rAAV vectors). This advance has enabled us to rigorously evaluate systemic administration of our most promising microdystrophin construct as a prospective treatment for DMD in the dystrophin-/utrophin- double-knockout mouse. This model experiences severe and progressive muscular dystrophy that shortens lifespan by 75% compared with wild-type and mdx mice. We observed that systemic administration of rAAV6-microdystrophin vectors restores dystrophin expression in important skeletal muscles body-wide and throughout the mouse heart. Restoration of dystrophin expression considerably reduced key indices of muscle disease (such as muscle fiber degeneration) in hindlimb and diaphragm muscles. Treatment also enhanced the force-producing capacity, and resistance to contraction-induced injury of limb and respiratory muscles, and dramatically reduced serum-based indicators of body-wide muscle fiber degeneration. Importantly, we have established that these effects of intravenous rAAV6-microdystrophin delivery preserve the muscle mass and physical mobility of dystrophic mice, and considerably extend their lifespan. These data provide evidence that systemic administration of AAV vectors is a promising strategy to achieve whole body treatment of pathology associated with muscular dystrophy, and as such may lead to a treatment for DMD and other severe neuromuscular disorders.
 

2) Ex Vivo Gene Therapy for Duchenne Muscular Dystrophy: Lentiviral Vectors, Exon Skipping and PhiC31 Integrase Approaches

Simon P. Quenneville, Pierre Chapdelaine, Joel Rousseau, Jacques P. Tremblay Human Genetic, CRCHUL, Quebec, QC, Canada

Duchenne muscular dystrophy is the most severe muscular dystrophy. It is caused by the absence of dystrophin in muscle fibers. This absence lead to increased muscle damage, loss of muscle mass, loss of strength, respiratory and heart failure. This disease as currently no treatment. Myogenic cells transplantation is a possible cure for Duchenne muscular dystrophy. However, allogeneic graft success relies on the use of efficient but toxic immunosuppressive drugs. The use of these drugs is a major problem that could be solved by the use of ex vivo gene therapy. This method consists in genetically modifying patient myoblasts before their auto-transplantation. In this study, a viral (lentiviral) and a non viral (integrase PhiC31) method were tested to perform the genetic modification. Therapeutic sequences included microdystrophin, dystrophin and exon skipping cassettes.
Co-transfection (nucleofection) of a PhiC31 integrase and a dystrophin expressing plasmid was used to perform the genetic modification. We have also generated eGFP, eGFP-microdystrophin and exon skipping lentiviral vectors with CMV and MCK promoters. Following in vitro modification of dystrophic cells (mdx mice derived) and normal human myoblasts, the cells were engrafted into mdx and SCID mice muscles.
The nucleofection led to a stable expression of the full length dystrophin. This was the biggest expression cassette ever stabilized in primary cultured human myoblasts. Following transplantation, the dystrophin expression was observed into a muscle, leading to the apparition of the dystrophin associated proteins a-sarcoglycan and b-dystroglycan. However, this method is not very efficient. We have also generated eGFP and eGFP-micro-dystrophin expressing lentiviral vectors under the control of the CMV and MCK promoters. Following in vitro infection of human myoblasts, the cells were engrafted into SCID mice muscles. Both transgenes were expressed into the muscles one month after the engraftment. The expression of microdystrophin also led to the apparition of a-sarcoglycans in the dystrophic model mice. We have also used a lentiviral vectors coding for an exon skipping cassette allowing us to skip the exon 51. In vivo experiments in SCID mice as shown that it is possible to express a quasi dystrophin protein with cells originating from a DMD patient. This work indicates that ex vivo gene therapy is a possible approach to treat Duchenne muscular dystrophy.
 

3) Recombinant AAV Gene Delivery of Follistatin for Muscle Enhancement in Models of Muscular Dystrophy

Liza Rizo, Chris Shilling, Amanda Haidet, Priya Umapathi Umapathi, Zarife Sahenk, Jerry R. Mendell, Brian K. Kaspar Gene Therapy and Neuromuscular Research, Columbus Children's Research Institute/The Ohio State University, Columbus, OH

Objective: To determine the efficacy of recombinant adeno-associated virus (rAAV) delivering follistatin (FS), a potent inhibitor of myostatin, to a mouse model for limb-girdle muscular dystrophy and wild-type animals.
Background: LGMD2D is a debilitating muscle disease of children and young adults. There is no proven treatment to delay the disease progression. Inhibition of myostatin, a negative growth modulator for muscle, can functionally improve normal or dystrophic muscle. This has been observed across species lines, through genetic manipulation, naturally occurring mutations or protein-specific antibodies. We have employed a novel gene therapy approach using a rAAV vector carrying a FS transgene.
Design/Methods: Four-week-old wild-type, mdx, or alpha-sarcoglycan deficinet mice were injected bilaterally into the quadriceps and tibialis anterior muscles with 1x10e10 viral genomes of rAAV1-FS and evaluated for muscle strength and endurance using hindlimb and forelimb grip strength as well as Rotarod. At age 120 days, gross muscle evaluation and analysis of muscle weight, fiber number, and cross-sectional area (CSA) on H&E sections was performed with the addition of a FS ELISA assay.
Results: We found increased serum FS levels accompanied by a local and remote increase in muscle mass in treated animals (p<0.05). Muscle hypertrophy was observed in rAAV-FS mice versus controls. Motor function tests showed statistically significant improvements in muscle strength in FS-treated mice.
Conclusions/Relevance: This data supports a therapeutic effect in muscular dystrophy by myostatin inhibition. This approach represents a clinically applicable gene therapy method to enhance muscle mass and function in muscular dystrophy with potential for treatment of other muscle diseases. No toxicity was encountered. The FS transgene could also be used to complement gene replacement therapy. For example, adding muscle mass in patients with more advanced dystrophies could improve their candidacy for gene replacement therapy.
 

4) C-Terminal Truncated Microdystrophin Recruits Dystrobrevin and Syntrophin to the Dystrophin-Associated Glycoprotein Complex and Reduces Muscular Dystrophy in Symptomatic Utrophin/Dystrophin Double Knock-Out Mice

Yongping Yue, Mingju Liu, Dongsheng Duan Molecular Microbiology and Immunology, University of Missouri, Columbia, MO

C-terminal truncated (C) microdystrophin is being developed for Duchenne muscular dystrophy (DMD) gene therapy. Encouraging results have been achieved in the mdx mouse model. Unfortunately, mdx mice do not display the same phenotype as human patients. Evaluating C microdystrophin in a symptomatic model will be of significant relevance to human trial. Utrophin/dystrophin double knock-out (u-dko) mice were developed to mold severe dystrophic changes in human patients. In this study we evaluated therapeutic effect of the R4-R23/C microdystrophin gene (R4/C) after serotype-6 adeno-associated virus (AAV-6)-mediated gene transfer in neonatal u-dko muscle. At two months after gene transfer, percentage of centrally nucleated myofiber was reduced from 89.2% to 3.4% and muscle weight was normalized. Furthermore, we have demonstrated for the first time that C microdystrophin can eliminate interstitial fibrosis, macrophage infiltration and restore dystrobrevin and syntrophin to the dystrophin-associated glycoprotein complex. Interestingly neuronal nitric oxide synthase was not restored. The most impressive results were achieved in muscle force measurement. Neonatal gene therapy increased twitch and tetanic specific force. It also brought the response to eccentric contraction-induced injury to the normal range. In summary, our results suggest that the R4/C microgene holds great promise in preventing muscular dystrophy.
 

5) A Pseudo-Exon Derived from an Intronic Insertion Is Responsible for Duchenne-Like Muscular Dystrophy in the Welsh Corgi Dog

Bruce F. Smith, Joseph N. Kornegay Scott-Ritchey Research Center, Auburn University, Auburn, AL; College of Veterinary Medicine, University of Missouri, Columbia, MO

Duchenne Muscular Dystrophy (DMD) is the most common X-linked inherited disease, with an incidence of 1 in 3500 male births. This disease is relentlessly progressive and usually fatal by early adulthood. A wide range of mutations in the dystrophin gene have been characterized for DMD, as well as the less severe form, Becker's Muscular Dystrophy. DMD presents gene therapy challenges due to the size of the gene and resulting cDNA and the wide variety and complexity of the mutations involved. Animal models for DMD have been described in the mouse, cat and dog. The disease in mice and cats is significantly less severe than that seen in humans, while the canine disease, characterized by muscle hypertrophy, followed by muscle loss, fibrosis and death, closely mimics disease progression in affected humans. As is expected from the frequency of mutation in the human population, DMD has been recognized in several breeds of dogs including the Golden Retriever, German Short-Haired Pointer, Rottweiler, Labrador Retriever, West Highland White and Welsh Corgi. Where the mutation has been determined, each breed has demonstrated a new mutation. Here we report the identification of the mutation responsible for DMD in the Welsh Corgi. Affected dogs can be recognized at, or shortly after, birth by increased serum creatine kinase levels. These dogs show progressive muscle atrophy and fibrosis, stunted growth, contractures and consequent debilitation. However, several of the affected males have lived to sexual maturity. Muscle biopsies from affected Corgis show that most fibers remain unstained for dystrophin, while rare fibers show dystrophin staining using a variety of antibodies. Sequence analysis of the cDNA indicates a 166 base insertion between exons 13 and 14, based on the human exon structure. The inserted sequence shows a high degree of sequence similarity to a canine LINE-1 element. Sequence analysis of intron 13, which is approximately 25kb, indicates that there is an insertion in the intron. The insertion is immediately downstream of an AG dinucleotide pair and contains the 166 bases seen in the cDNA followed by a GT dinucleotide pair and additional inserted sequence. Thus the insertion utilizes a putative intronic 3'splice acceptor site and a 5' splice donor to form a novel exon, which is spliced into the mature mRNA. An in-frame stop codon in this novel exon results in a truncated dystrophin protein, which is non-functional. Staining of occasional fibers with antibodies to epitopes located downstream of this insertion indicates that this exon may be skipped or that alternative splicing and/or promoter use may produce a product. This large animal model, with its defined mutation, will be useful in a variety of ways, including the use of gene repair approaches to skip the inserted exon, studies of spicing mechanisms in dystrophin, and studies of other gene therapy approaches.
 

6) Antisense Oligonucleotide Therapy for Duchenne Muscular Dystrophy: From Cell Culture to Clinical Trial

Jignya Ashar, Terence Partridge, Qi L. Lu MocColl-Lockwood Laboratory for Muscular Dystrophy Research, Carolinas Medical Center, Charlotte, NC; Research Center for Genetic Medicine, Children's National Medical Center, Washington, DC

Duchenne muscular dystrophy (DMD) results from non-sense or frame-shifting mutations in the dystrophin gene. The majority of these mutations can be corrected by removal of one extra exon to create shortened, but in-frame transcripts and produce at least partially functional proteins. Earlier studies showed that antisense oligonucleotide (AON)-mediated splicing alteration can achieve specific skipping of targeted exon, providing the potential for the treatment of DMD. This promise was firmly established when we demonstrated that AON can specifically remove the mouse dystrophin exon 23 containing a nonsense point mutation and restore the functional levels of dystrophin protein in muscle via intramuscular administration. More recently, we further demonstrated that systemic delivery of AON can effectively restore dystrophin expression in body-wide muscles. These data have prompted much of the current enthusiasm for clinical trials with AONs. We also demonstrated that modifications of AON chemistry are critical for achieving therapeutical effect by antisense therapy. Morpholinos is one of the most effective chemistry for inducing functional levels of dystrophin in bodywide skeletal muscles with improved force generation. We have been examining several chemistries with various delivery systems using mdx mouse as an in vivo model system to search for more effective chemistry and clinically applicable delivery approaches to achieve long-term restoration of dystrophin expression. Individual human dystrophin exon has been selected for systematic screening of AON sequences to maximize the potential of antisense effect as a preparation for clinical trials. We are also developing animal models to select AON sequences for targeting individual human dystrophin exon and to examine the therapeutic capacity of exon skipping in vivo. In conclusion, our results have provides realistic hope for the treatment of a majority of DMD patients.
 

7) Development of Recombinant Novel Adeno-Associated Viral (rAAV) Vectors Encoding Optimised Microdystrophin cDNAs for Duchenne Muscular Dystrophy (DMD)

Takis Athanasopoulos, Ian Graham, Capucine Trollet, Helen Foster, Norma Perez, Vanessa Hill, Phillippe Moullier, George Dickson Biochemistry, Centre for Biomedical Sciences, Royal Holloway University of London, Egham, Surrey, United Kingdom; Gene Therapy Lab, Genethon, Evry, Paris, France; Laboratoire de Therapie Genique, Inserm, Nantes, France

Duchenne muscular dystrophy (DMD) is a lethal genetic muscle disorder affecting 1:3500 male individuals, caused by recessive mutations in the dystrophin gene. The size of the gene (2.4Mb) and mRNA (14kb) in addition to immunogenicity problems and inefficient transduction of mature myofibres by currently available vector systems are formidable obstacles to the development of gene therapy. AAV vectors overcome many of the problems associated with other vectors but accommodate limited transgene capacity (<5kb), allowing only truncated versions of dystrophin (microdystrophin) to be packaged. More than 9 AAV vector serotypes have been identified to date with certain serotypes(1, 5-9) displaying more favourable tropism in transducing muscle compared to the traditionally used AAV2. Human microdystrophin (mdys) cDNAs (<3.8 kb) rationally designed in our lab based on patient and preclinical data were packaged in AAV-based vector cassettes, under the control of CMV or Spc512 muscle synthetic promoter, effectively expressed mdys (CMV>Spc512), restored DAP complex and inhibited myofibre degeneration after local intramuscular administrations in nude/mdx and mdx mice. Expression of microdystrophins using e.g. an AAV1CMVhmdys vector was observed at high levels via i.m (>90%), i.p. (20-25%), but not via systemic i.v. route of administration. However, certain issues including systemic delivery, optimization of mdys cDNA constructs, AAV transduction efficiency, safety and immunogenicity while retaining species and tissue (muscle) specificity, still remain to be fully elucidated.
Here we report on production of codon/sequence optimised, transcriptionally enhanced, species-specific (human, canine and murine) microdystrophin cDNA cassettes (based on 3788 and R4-23 published sequences) incorporating either constitutive or muscle-specific promoter elements. These cassettes offered 3-5 fold increase in expression in comparison to conventional configurations. Incorporation of Kozak/WPRE elements for transcriptional enhancement, packaging of the cassettes into recombinant AAV vectors of novel alternative serotypes (6, 7, 8 9) by pseudotyping and AAV-stealth approaches have been envisaged. Resulting rAAV6 and rAAV8 pseudotyped vectors encoding species-specific comdys have been produced at titers>10E12 vg/mL and are currently screened utilising local and systemic routes of administration in preclinical models of the disease.
 

8) Enhanced Level of Gene Correction Mediated by Oligonucleotides Containing CpG Modification in the mdx Mouse Model for Duchenne Muscular Dystrophy

Carmen Bertoni, Arjun Rustagi, Thomas A. Rando Neurology, Stanford University, Stanford, CA; Neurology Service and GRECC, VAPAHCS, Palo Alto, CA

Single point mutations and large deletions of the dystrophin gene are responsible for Duchenne muscular dystrophy (DMD) a severe neuromuscular disorders characterized by complete absence of dystrophin expression in skeletal muscles. Gene editing mediated by single stranded oligonucleotides (ssODNs) represents an appealing option to treat the disease since it has the potential to treat both single point mutations as well as deletions that cause frame shift of the dystrophin mRNA. The major limitation of the technology has been so far the low level of genomic correction detected in muscle cells.
We have focused on the development of new vectors capable to activate specific repair mechanisms and capable of directing the repair process specifically on the sequence of the genomic DNA targeted for correction. The methyl binding protein 4 (MBD4) takes an active role in the base excision repair mechanism and is highly expressed in muscles. In addition to containing a binding site for the methylcytosine, MBD4 contains also a specific glycosilase capable of recognizing a T to G transversion at CpG sites and direct the conversion of the thymine into methylcytosine. CpG modifications were introduced on the mutating base of the targeting oligonucleotide in the attempt to mimic a deamination of methylcytosine which results in the activation of MBD4.
All the studies were conducted using the mdx mouse as a model for DMD. This strain contains a stop codon in exon 23 of the dystrophin gene that is responsible for the absence of dystrophin protein in skeletal muscles. As a target for the single base substitution we have chosen the splice boundary of exon 23 of the mouse dystrophin gene in order to induced exon skipping to bypass the nonsense mutation and induce expression of internally deleted but functional dystrophin proteins. We have designed ssODNs complimentary to the coding or the non-coding strand of the donor site of exon 23. CpG modifications were introduced at the targeted base. The ability of these modified ssODNs to increase gene repair was studied in muscle cells both in vitro and in vivo. The level of dystrophin protein expression was significantly increased by the use of ssODNs containing CpG modifications on the targeting base. Studies conducted on muscle cells in culture demonstrate up-regulation of MBD4 mRNA and the activation of the base excision repair mechanism through which MBD4 acts, demonstrating the specificity of the repair process recruited by the ssODNs. Correction of the dystrophin gene was shown to occur at the genomic level and was stable over prolonged periods of time. In muscle cells in culture, restoration of dystrophin expression was analyzed at the protein level by western blot and immunostaining analysis and at the mRNA level by RT-PCR. In vivo analysis also showed restoration of dystrophin in skeletal muscles of injected mice by immunostaining. These studies demonstrate that the efficacy of oligonucleotide-mediated gene correction can be increased by improving oligonucleotide design.
 

9) Phi C31 Integrase System Enhances Dystrophin Gene Expression in Skeletal Muscle of Mouse Models for Duchenne Muscular Dystrophy

Carmen Bertoni, Sohail Jarrahian, Thurman M. Wheeler, Yining Li, Eric C. Olivares, Michele P. Calos, Thomas A. Rando Neurology, Stanford University, Stanford, CA; Genetics, Stanford University, Stanford, CA

Duchenne muscular dystrophy (DMD) is caused by lack of dystrophin expression in skeletal muscles. To be effective, gene therapy approaches to DMD need to target a large number of fibers in the muscle, and the distribution of dystrophin through the fiber length needs to be sufficient to prevent fiber degeneration. Plasmid-based gene therapies have been shown to be a valid approach to the treatment of a variety of disorders including DMD. Among the limitations of this technology are the restricted distribution of plasmid following direct intramuscular injection, the challenge of achieving widespread plasmid delivery by systemic injection, and the loss of extrachromosomal plasmid over time. In order to address the latter issue, which could prove to be a major limitation of therapeutic efficacy in DMD, we have studied the effects of targeted plasmid integration using a phage integrase (Phi C31) that has been shown to mediate the integration of suitably modified plasmids into the mammalian genome. Using a luciferase expression plasmid, we were able to monitor plasmid gene expression non-invasively in living mice by bioluminescence imaging (BLI). Tibialis anterior muscles of wild-type animals were injected with a plasmid carrying a luciferase reporter gene under the control of the CK6 muscle specific promoter and a fC31 integrase attB site. One group of muscles was co-injected with an equal amount of a CMV-driven integrase-expressing plasmid (pCSI) to direct site-specific integration, while contralateral muscles received empty vector (pCS). All muscles were subjected to electroporation to achieve a high level of plasmid delivery, and expression was followed using BLIS. Shortly after delivery, the level of gene expression obtained in muscles that had received pCSI was 5- to 10-fold higher than in muscles receiving pCS. This increase in the gene expression level was not due to differences in delivery efficiency and was maintained for up to 2 years after injection.
Co-injection of an integrase plasmid resulted in up to 10-fold higher levels of sustained luciferase expression, and we demonstrated a direct correlation between levels of expression and plasmid integration. Likewise, dystrophin expression in mdx muscle was enhanced by co-injection with an integrase plasmid. Using a combination of dystrophin and luciferase plasmids, we were able to follow the functional benefit of dystrophin expression in the dystrophic muscle. The expression of dystrophin slowed the loss of luciferase expression associated with muscle degeneration, and that protection was even greater when the dystrophin plasmid was co-injected with the integrase plasmid. In the presence of integrase, dystrophin expression was enhanced in individual fibers and was detectable along the entire length of a subset of fibers. Those fibers were protected indefinitely. These data demonstrate the importance of both the level and the distribution of dystrophin expression to achieve therapeutic efficacy and that the efficacy can be enhanced by targeted plasmid integration.
 

10) Gene Therapy for Duchenne Muscular Dystrophy Using a Gutted Adenovirus Expressing Utrophin

Jatinderpal R. Deol, Renald Gilbert, Gawiyou Danialou, Mylene Bourget, Joon-Shik Moon, Basil J. Petrof, Josephine Nalbantoglu, George Karpati Neuromuscular Research, Montreal Neurological Institute, Montreal, QC, Canada; Genomics and Gene Therapy Vectors, Biotechnology Research Institute, Montreal, QC, Canada; Department of Neurology, UMDNJ-New Jersey Medical School, Newark, NJ; Respiratory Division, Meakins Christie Laboratories, Montreal, QC, Canada

Duchenne muscular dystrophy (DMD) is characterized by a mutation in the gene encoding a sarcolemmal protein, dystrophin (dys) and the deficiency of dys leads to progressive loss of muscle fibers. Gutted adenovirus (AdV) vectors are less immunogenic than their predecessors and have the capacity of carrying large DNA inserts such as the full-length dys cDNA (13 kb). The use of gutted AdV vectors encoding full-length dys leads to significant improvement of the dystrophic phenotype of the mdx mouse, an animal model of DMD. However, dys behaves as a neoantigen in genetic dys deficiency states such as the mdx and it causes deleterious immune responses in the treated muscles. An alternative approach would be to use utrophin (utr), a functional homologue of dys, which is normally present only at the sarcolemma of the neuromuscular and myotendinous junctions. Therefore, utr is not expected to behave as a neoantigen in dys-deficient states. In fact, transgenic mdx mice expressing extrasynaptic utr in muscle fibers have shown functional and histological improvement. Previous findings have also shown utrophin, when expressed via a first generation adenovirus to be sufficient and adequate in replacing dystrophin at the sarcolemma and preventing the symptoms of DMD. Thus, in an attempt to minimize the vector immunogenicity and to harness the therapeutic potential of utr, we created a fully deleted AdV encoding full-length utrophin. The vector was administered to cohorts of mdx neonates (2-4 day old) and adults (5 to 7 week old) at doses of 1.45 x 10¹⁰ and 4.35 x 10¹⁰ virus particles respectively into the tibialis anterior (TA) muscles. In neonates at 10 days post-injection (pi), the mean number of extrasynaptic utr+ fibers in vector vs control-injected TA was 1596 vs 114, corresponding to 58 % transduction level. At 60 days pi, the level of transduction was found to be near 52% corresponding to approximately 794 utr+ fibers. The levels in adults at 10 days pi were 685 and 112 for vector and control injected TAs respectively, corresponding to a 23% transduction level. At the 10 day timepoint, there was a 14- and 6- fold increase in utr expression over control levels in neonates and adults. In adults at 30 days pi, the mean number of utr + fibers was 770 corresponding to 35% transduction level. However, that number dropped substantially to an average of 80 fibers at 60 days pi and remained between 80 and 120 up to the 180 days pi. At no time point were anti-utrophin antibodies detected. The decline of the initial high utr transduction of the TA muscles associated with time may be due to the fact that the initial extrasynaptic utr level is not sufficiently high to prevent the natural progression of the dystrophic phenotype. Further augmentation of the initial utr transduction should be helpful in order to completely and functionally compensate for dys.
 

11) Dystrophin Expression in Muscles of Duchenne Muscular Dystrophy Patients Following High-Density Injections of Normal Myogenic Cells

Jacques P. Tremblay, Daniel Skuk, Marlyne Goulet, Brigitte Roy, Pierre Chapdelaine, Raynald Roy, Francine Dugré, Jean-Pierre Bouchard, Jean-Guy Lachance, Michel Sylvain, Louise Deschênes, Hélène Senay Anatomy/Physiology, Laval Universite, Quebec, QC, Canada; Immunologie, CRCHUL, Quebec, QC, Canada; Departement de Microbiologie, CHUL, Quebec, QC, Canada; Departement Neuropediatrie, CHUL, Quebec, QC, Canada; Neurologie, Hopital de l'Enfant-Jesus, Quebec, QC, Canada; Service de Nephrologie, Hotel-Dieu de Quebec, Quebec, QC, Canada; Service de Microbiologie, Hotel-Dieu de Quebec, Quebec, QC, Canada

A clinical trial was conducted to test a new protocol of normal muscle-precursor cell (MPC) allotransplantation in skeletal muscles of Duchenne muscular dystrophy (DMD) patients. The protocol consisted on using high-density cell-injections as a method for cell delivery and a tacrolimus-based immunosuppression as a method to control acute rejection. Cultured MPCs obtained from one of the patient's parents were implanted in 0.25 or 1 cm3 of a Tibialis anterior in 9 DMD patients. MPC injections were placed 1 to 2 mm from each other, and a similar pattern of saline injections was done in the contralateral muscle. The patients were immuno-suppressed with tacrolimus. Muscle biopsies were performed at the injected sites 4 weeks later. In the biopsies of the cell-grafted sites, there were myofibers expressing donor's dystrophin in 8 patients. The percentage of myofibers expressing donor's dystrophin varied from 3.5% to 26%. Evidence of small-myofiber neoformation was observed in some patients. Donor-derived dystrophin transcripts were detected by RT-PCR in the cell-grafted sites in all patients. The protocol of immunosuppression was sufficient to obtain these results, although it is not certain whether acute rejection was efficiently controlled in all the cases. In conclusion, intramuscular allotransplantation of normal MPCs can induce the expression of donor-derived dystrophin in skeletal muscles of DMD patients, although this expression is restricted to the sites of MPC injection.
 

12) Muscle Specific and Efficient Transgene Expression Using the Fast and Slow Troponin I Promoters in Cell Culture

Marilyne Blain, Angela Kumar, Kenneth E. Hastings, George Karpati, Bernard Massie, Renald Gilbert Genomics & Gene Therapy Vectors, Biotechnology Research Institute, NRC, Montreal, QC, Canada; Neuromuscular Research Group, Montreal Neurological Institute, McGill University, Montreal, QC, Canada

Gene replacement therapy for muscular diseases, such as Duchenne muscular dystrophy, using viral vectors requires strong promoters with the ability to confer muscle specific expression of the therapeutic gene product. Because skeletal muscle consists of a mixture of slow and fast muscle fibers, an ideal promoter for such application should direct efficient expression in both muscle fiber types. The genetic elements regulating expression of the fast and slow isoforms of troponin I (TnI), an abundant muscle protein, are relatively well characterized. Theoretically, a combination of vectors expressing transgenes regulated by TnIfast and TnIslow enhancers/promoters could be used to deliver strong and muscle specific transgene expression in every skeletal muscle fiber of the body.
To evaluate the usefulness of the TnIfast and TnIslow enhancers/promoters for gene therapy, we have compared their strengths with the cytomegalovirus (CMV) and with the short (1.3-kb) murine muscle creatine kinase (MCK) promoters in cell culture. Plasmids expressing -galactosidase (-gal) regulated by MCK, CMV or TnIfast / TnIslow enhancers/promoters were constructed. The TnIfast construct includes the minimal promoter (200-bp) of the quail TnIfast gene linked to three copies (150-bp each) of that gene's first-intron enhancer element (IRE). The TnIslow construct includes the minimal promoter (100-bp) of the human TnIslow gene linked to that gene's upstream enhancer element of 160-bp (USE). Except for MCK, a small intron (100-bp) derived from SV40 was inserted between the promoter and -bal. 293A cells and C2C12 myoblasts were transfected with the different constructs and -gal expression was compared 48 hrs later. Analyses were also performed in culture of C2C12 myotubes 10 days after serum withdrawal.
In 293 cells, MCK-, TnIfast-, and TnIslow-driven expression was at least 1300 folds weaker than CMV. In non-differentiated myoblasts, TnIslow-driven expression was 3.5 and 15 times better than TnIfast- and MCK-driven expression, respectively. Both TnIfast and TnIslow enhancers/promoters provided high-level -gal expression in myotubes (35-40 % of CMV, and at least 10 times stronger than MCK).
Our data confirmed the excellent muscle specificity and strength of TnIfast and TnIslow enhancer/promoter elements. Their relative small size makes them very attractive for usage in vectors with limited insert capacity such as AAV. These excellent properties warrant further in vivo gene transfer studies using viral vectors.
 

13) Efficient and Selective Gene Transfer Directed to Muscle by Tropism-Modified Adeno-Associated Virus Vector

Chi-Yi Yu, Bing Wang, Zhong Wang, Zhongren Cao, Juan Li, Xiao Xiao Department of Orthopaedic Surgery, School of Medicine, University of Pittsburgh, Pittsburgh, PA

Duchenne muscular dystrophy (DMD) is a lethal genetic muscle disorder that affects one in 3500 males. Transfer of the therapeutic gene into the diseased muscles has great potential for the treatment of DMD patients. Gene delivery vehicles based on recombinant adeno-associated virus (rAAV) have been generated as promising vectors for the correction of genetic diseases. However, rAAV primarily transduces the liver after systemic administration, reducing its efficiency for gene transfer at skeletal muscle. The question of how to deliver the therapeutic genes into most of the diseased myofibers specifically becomes a challenge. Our goal is to develop an efficient and muscle-specific AAV vector for systemic gene delivery. In this study, we genetically incorporated the muscle-targeting peptide into AAV2 capsid and evaluated its efficiency and selectivity for muscle targeting.
In order to validate the targeting effect of the muscle-targeting peptide for AAV-mediated gene transfer, differentiated C2C12 myotubes were infected with modified and wild-type vectors harboring a luciferase reporter gene. The modified AAV2 viruses were able to transduce the differentiated C2C12 myotubes 2- to 3-fold better than wild-type AAV2. We further determined cell type specificity of the modified AAV2 targeting. Transductions of undifferentiated C2C12 myoblasts, HeLa cells, HepG2 cells, and 293 cells resulted in 97% ~ 99% decreases in reporter gene activities compared to the vector carrying a wild-type capsid. This indicated the modified AAV2 viruses significantly increased their selectivity for targeting. We next examined if heparin could act as a competitor to impact the transduction efficiency of the modified AAV2 in C2C12 myotubes since a primary receptor for AAV2 has been identified to be heparan sulfate proteoglycan. The result showed that the modified AAV2-mediated transduction of C2C12 myotubes was not affected by heparin. Significant gene transduction was maintained even in the presence of heparin when C2C12 myotubes were infected with the modified AAV2 vectors. Heparin dependence was also determined by the heparin-affinity column analysis. In contrast to wild-type AAV2 vector, peptide-modified AAV vector was substantially detected in the column wash, consistent with the cell-transduction studies.
Our work is the first time muscle tropism was increased through peptide-engineered AAVs. First, we demonstrated that enhanced transduction of myotubes is mediated by this muscle-targeting peptide. Second, peptide-engineering AAVs decreased their transduction in non-muscular control cell lines, such as HepG2 and 293 cells. Third, we found the modified AAV2 did not require the heparin-dependent mechanism for muscular targeting. In summary, this study demonstrates the ability of genetically engineered gene therapy vectors for selective delivery of genes to target muscle cells. These results make us believe the use of this muscle-targeting peptide might facilitate the generation of efficient and muscle-specific AAV vectors for systemic gene delivery in the future.
 

14) Histone Deacetylase Inhibitors Improve Gene Transfer To Mature Skeletal Muscle

Nancy Larochelle, Jatinderpal R. Deol, Paul Holland, George Karpati, Josephine Nalbantoglu Neuroimmunology, Montreal Neurological Institute, Montreal, QC, Canada

AdV-mediated gene transfer occurs inefficiently in adult skeletal muscle due to downregulation of the primary adenovirus receptor CAR during muscle maturation. We have shown previously that AdV-mediated gene transfer is enhanced in CAR transgenics, indicating that prior modulation of CAR expression results in very efficient AdV transducibility of mature skeletal muscle. We hypothesized that transient CAR expression could be achieved through activation of the transcription of the endogenous CAR gene using histone deacetylase (HDAC) inhibitors. HDACs are known to regulate gene expression via the modulation of chromatin structure by histone acetylation and deacetylation.
To assess the effect of histone deacetylase inhibitors on CAR expression and adenovirus-mediated gene transfer, mouse myoblasts (C2C12) were pre-treated with the HDAC inhibitor valproic acid (VPA) and infected with AdVCMVlacZ. Quantification of -galactosidase activity 24 hours post-infection revealed a 5-100 - fold increase in enzyme activity which depended on the multiplicity of infection (MOI). CAR expression, in cells treated with VPA, increased by a factor 3 as determined by Western blotting. VPA treatment had minimal effect on C2C12 myoblast transducibility by an Ad35 expressing lacZ, indicating that the effect is specific for the Ad5 receptor CAR. Similar treatment of primary myoblast cultures derived from a patient afflicted with Duchenne muscular dystrophy (DMD) also produced a comparable 5-300 - fold increase in -galactosidase activity when the VPA-treated cells were transduced with AdCMVlacZ at various MOI.
To assess in vivo effects, adult normal and dystrophin-deficient (mdx) mice were treated with VPA by intraperitoneal injection (ip). Gene transfer was evaluated 7 days after intramuscular injection of AdCMVlacZ. In normal mice, no difference was seen in the number of transduced fibers and in the -galactosidase activity between VPA-treated animals and controls. On the other hand, a 2-fold increase was detected in mdx mice pretreated with VPA compared to non-pretreated mice. This augmentation in transduced fibers in mdx muscles correlated with an increase in CAR transcript (up to 7-fold) as determined by qRT-PCR.
The HDAC inhibitor valproic acid renders muscle cells more susceptible to adenovirus-mediated gene transfer. This may have future implications for muscle-directed gene therapy in muscular dystrophies.
 

15) rAAV2/9 Mediated Gene Delivery of Acid -Glucosidase Corrects the Cardiac Phenotype in a Mouse Model of Pompe Disease

Christina A. Pacak, Cathryn Mah, Kerry O. Cresawn, Melissa A. Lewis, Sean Germain, Barry J. Byrne Molecular Genetics & Microbiology, University of Florida, Gainesville, FL; Cell & Molecular Therapy, UF, Gianesville, FL; Pediatric Cardiology, UF, Gianesville, FL

The long term goal of this project is to develop a clinically relevant gene therapy approach for the treatment of Pompe Disease. Pompe Disease is a form of muscular dystrophy and metabolic myopathy caused by mutations in the acid alpha glucosidase (GAA) gene. An insufficient amount of GAA leads to the accumulation of glycogen in lysosomes and consequent cellular dysfunction. Cardio-respiratory failure typically occurs in the early onset patients within the first year of life. We have characterized the cardiac phenotype in our mouse model (gaa-/-) at various ages. Through ECG analysis we observe a shortened PR interval by 3 months of age (gaa-/-33.41+1.35ms, cont 44.95+1.58ms) mimicking the conduction phenotype in the human Pompe population. Abnormal amounts of glycogen are observed in lysosomes as demonstrated by the periodic acid shift (PAS) stain. MRI analysis shows a decrease in stroke volume (SV)(gaa-/-36.13+1.19ul, cont 51.84+3.59ul) and a decrease in cardiac output (CO)(gaa-/-7.95+0.26ml/min, cont 11.40+0.79ml/min) at 3 months and an increase in mass (gaa-/-181.99+10.7mg, cont 140.79+5.12mg) by 12 months. This model of cardiac dysfunction is being used to develop a cardiac gene delivery technique which can be applied to many genetically inherited cardiomyopathies. Previously, we have shown that intra-venous (IV) delivery of recombinant adeno-associated virus type 1 (rAAV2/1) pseudotype capsid carrying the CMV-hgaa construct to 1 day old gaa-/- neonates restores GAA activity in mice. Also, LacZ transgene delivery using the IV administration route and rAAV2/9 pseudotype capsid resulted in 200 fold higher levels of expression in cardiac tissue than rAAV2/1. Additional experiments showed transduction following delivery to adults. We have now combined rAAV2/9 with the clinically relevant IV administration route in order to deliver the human GAA (hgaa) gene to gaa-/- mice. Neonates treated with rAAV2/9-CMV-hgaa at a range of doses (4x10⁵vg 4x10⁸vg 4x10¹⁰vg) have demonstrated sustained PR interval correction (39.38+2.42 ms). PAS stains as well as NMR analysis have shown less glycogen accumulation in cardiac tissue of treated gaa-/- mice as compared to untreated mice. MRI analysis shows an increase in SV and CO. Adult gaa-/- mice have also been treated using this strategy and are being assessed in order to determine if we have the ability to reverse the effects of Pompe Disease in mice already presenting the cardiac phenotype. While the focus of this project is on correction of the cardiac phenotype of Pompe Disease, our systemic delivery route, use of the CMV promoter and the fact that GAA is a secreted enzyme all promote correction throughout the body. GAA activity is observed in other tissues of treated mice including skeletal muscles and liver. These studies have demonstrated the ability of rAAV2/9 to be administered systemically using the IV delivery route, transcend the vasculature, transduce tissues throughout the body and ultimately prevent presentation of the cardiac phenotypes of Pompe Disease.
 

16) Correction of Ventilation in Glycogen Storage Disease Type II Mice after Gel-Mediated Delivery of Adeno-Associated Virus Serotype 1 Vectors

Cathryn Mah, Lara R. DeRuisseau, Christina A. Pacak, Melissa A. Lewis, David D. Fuller, Barry J. Byrne Pediatrics, Div of Cell and Molecular Therapy, and Powell Gene Therapy Center, University of Florida, Gainesville, FL; Physical Therapy, University of Florida, Gainesville, FL

Glycogen storage disease type II (GSDII) is an autosomal recessive disorder caused by a lack of functional acid -glucosidase (GAA) and results in massive storage of glycogen in lysosomal compartments. It is characterized by progressive skeletal muscle weakness and affected patients suffer severe respiratory insufficiency, oftentimes requiring mechanical ventilation. Previously, we demonstrated that a novel gel-mediated method of delivery of recombinant adeno-associated virus serotype 1 (rAAV2/1) vector to diaphragm in a mouse model of GSDII (Gaa^-/-) could restore therapeutic levels of diaphragmatic GAA enzymatic activity with concomitant clearance of glycogen in vivo. We have further characterized the effects of this treatment method on ventilatory function. Similar to the GSDII patient population, we demonstrate in Gaa^-/- mice that there is an age-related progressive weakening of diaphragm contractile strength that is accompanied by ventilation impairments. We administered 1x10¹¹ particles rAAV2/1-CMV-GAA to diaphragms of Gaa^-/- mice at 3, 9, and 21-mos of age via the gel method. Diaphragm contractile strength and respiratory function were assessed by measuring force-frequency relationships and by barometric whole-body plethysmography, respectively. For plethysmography, ventilation was measured in conscious, unrestrained mice under conditions of normoxia (F_IO2:0.21, F_ICO2: 0.00) and hypercapnia (F_IO2: 0.21, F_ICO2: 0.07) to assess the extended range of ventilatory capacity. In mice treated at 3 mos of age, we show a significant improvement in contractile force at 6 mos (peak force of 24.833.31 vs 16.53+0.74 N/cm²) that is sustained through 1 yr (21.59+1.59 vs 13.94+1.15 N/cm²) of age as compared to age-matched untreated controls. Under conditions of normoxia, the ratio of minute ventilation (V_E; mL/min) to expired CO₂ (V_E/VCO₂) (18.65+0.73 vs 13.3+0.74), and peak inspiratory flow (mL/sec) (4.11+0.17 vs 3.21+0.29) were significantly improved (p<0.05) in mice treated at 3 mos of age and tested at 6 mos as compared to untreated controls. However, these improvements did not persist to 1 yr of age. In mice treated at 9 and 21 mos of age, there was significant improvement in contractile function in treated diaphragms 3 mos post-treatment (peak force of 21.28+1.49 at 1 yr and 17.21+0.29 N/cm² vs 12.71+0.94 N/cm² at 2 yrs of age, respectively) as compared to age-matched untreated controls. In all rAAV2/1-treated mice, minute ventilation and peak inspiratory flows were significantly improved during hypercapnia. These results demonstrate that gel-mediated delivery of rAAV2/1 vectors improve ventilatory function in a mouse model of muscular dystrophy.
 

17) Novel Tissue-Specific Regulatory Cassettes Direct High-Level Transgene Expression in Skeletal and Cardiac Muscle

Maja Z. Salva, Charis L. Himeda, Phillip Tai, Eiko Nishiuchi, Eric E. Finn, James M. Allen, Paul Gregorevic, Michael J. Blankinship, Leonard A. Meuse, Jeffrey S. Chamberlain, Stephen D. Hauschka Bioengineering, University of Washington, Seattle, WA; Biochemistry, University of Washington, Seattle, WA; Neurology, University of Washington, Seattle, WA

Systemic delivery of rAAV6 vectors can achieve efficient transduction of the entire striated musculature, making this an attractive strategy for gene therapy of Duchenne Muscular Dystrophy. However, this delivery method also transduces cells in many non-muscle tissues, which may cause therapeutic problems including toxicity and a transgene-directed immune response. These problems could be avoided by limiting transgene expression through the use of muscle-specific promoters. Unfortunately, the expression levels of such promoters following systemic delivery of rAAV vectors are lower than viral promoters in most skeletal muscles and negligible in cardiac muscle. The goal of this project is to design regulatory cassettes that drive high levels of skeletal and cardiac muscle-specific transgene expression while also being sufficiently short (<800bp) to fit into rAAV vectors containing microdystrophin cDNA (3.8-kb). Our lab has developed a series of muscle-specific regulatory cassettes based on the enhancer and promoter of the murine muscle creatine kinase (MCK) gene. The CK6 cassette, previously thought to be the strongest modification, has higher activity than the wild type cassette in skeletal muscle, but very low activity in cardiac and diaphragm muscle. We have designed new cassettes to increase expression, and their activities have been evaluated in various muscles and non-muscle tissues following systemic delivery of rAAV6 vectors expressing a human placental alkaline phosphatase reporter gene. First, we designed a 570-bp MCK-based cassette, CK7, which exhibited activity levels similar to CK6 in quadriceps, a predominantly fast-twitch muscle, but had significantly higher activity in cardiac muscle, diaphragm, and soleus, a predominantly slow-twitch muscle. To further improve expression, especially in cardiac muscle, we added a 190-bp enhancer from the murine alpha-myosin heavy chain gene (MHC) to the CK7 cassette to produce MHCK7, a 770-bp cassette that has the highest overall activity. When compared to CK6, the activity of MHCK7 was about 400-fold higher in cardiac muscle and 10-fold higher in soleus, which was equivalent to the activity of the CMV promoter. Staining for AP activity in cross-sections of both muscles revealed strong expression in virtually all fibers. The activity in diaphragm was about 40-fold higher, but was still considerably lower than the CMV promoter, with only about 20% of the fibers staining positive. Finally, the activity in quadriceps muscle was equivalent to CK6. The activity of MHCK7 in kidney, aorta, brain, testes, and intestine was at background levels, while a very slight increase in activity, about 50-200-fold lower than in muscle, was detected in liver, spleen, and lung. Based on these results, the MHCK7 cassette is currently being tested with respect to its capacity for driving microdystrophin expression in mdx mice following intramuscular and systemic injections of rAAV6 vectors.
 

18) Non-Invasive Measurement of Maximal Strength in the Normal and Dystrophic Dog Quadriceps Using Magnetic Stimulation of the Femoral Nerve

Frederick J. Balzer, Martin K. Childers, Janet R. Bogan, Daniel J. Bogan, Joe N. Kornegay, Hansell H. Stedman Surgery, University of Pennsylvania School of Medicine, Philadelphia, PA; Physical Medicine and Rehabilitation, School of Medicine University of Missouri-Columbia, Columbia, MO; Medicine and Surgery, College of Veterinary Medicine University of Missouri-Columbia, Columbia, MO

FJB and MKC contributed equally to this study. We have previously reported in detail the first gene transfer method to achieve virtually 100% myofiber transduction in proximal limb muscles of the dog - thus establishing the feasibility of high efficiency somatic gene transfer to striated muscle in a large mammal. Our group is currently undertaking to study the efficacy of therapeutic gene delivery in a canine model for Duchenne Muscular Dystrophy. Towards this end, our group has developed a novel, completely non-invasive, non-volitional assessment of proximal (quadriceps) muscle strength in the dystrophic dog using magnetic stimulation of the femoral nerve. Using a computer controlled 1.4 Tesla electro-magnetic coil capable of inductively triggering isolated single twitches up to complete fusion and sustained supramax titanic contraction, we initially studied quadriceps force produced by inductive magnetic stimulation of the femoral nerve in normal dogs with the result that supramaximal stimulation is rapidly achieved and well tolerated in lightly sedated dogs. Following this, we repeated the same experiment on a representative set of dystrophic dogs from the GRMD colony. Again the magnetic stimulation was well tolerated in this context, importantly in the oldest dog studied, a lightly sedated 18 month old GRMD male. Statistical analysis of the quadriceps tetanic force data for normal and GRMD dogs reveals that the general approach is highly reproducible. The average 8 month old GRMD dog tested had a maximal quadriceps force of 1.79 N/kg body weight, only 46% that of even the youngest normal dogs tested (3.91 N/kg at 7 weeks). In contrast, the previous tests of relative force showed that GRMD dogs doubled their strength in the TA/EDL muscle group from 3 to 6 months and thereafter maintained approximately 100% of the value found in the youngest normal dogs tested. Importantly, for the quadriceps force as measured by the present systems, the left versus right limb correlation is even better than that previously observed for tibiotarsal extension, and can be assessed in younger dogs. Based on our current experience we anticipate straightforward extrapolation of this approach to the measurement of quadriceps force in pups at 4 weeks, providing baseline information before the onset of clinically overt muscle weakness. We anticipate that the use of this test sequence in the DMD large animal model will allow us to define the response to regional gene therapy in terms of clinically relevant, statistically robust efficacy endpoints that are internally consistent and replicative.
 

19) Increased Transgene Expression of Dystrophin in mdx Muscle by RNAi-Mediated Silencing of Calpain Expression

Makiya Nishikawa, Kazuhiro Hirata, Kazuya Machida, Yuki Takahashi, Katsutoshi Yuasa, Shin'ichi Takeda, Yoshinobu Takakura Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan; Faculty of Pharmacy, Musashino University, Nishitokyo, Japan; Department of Molecular Therapy, National Institute of Neuroscience, NCNP, Kodaira, Japan

Plasmid DNA-based dystrophin gene transfer can be a way to cure Duchenne muscular dystrophy (DMD) at the molecular level. However, there are several technical difficulties in DMD gene therapy. Although intravascular delivery of naked plasmid DNA solves some of the problems associated with the delivery of dystrophin gene to widespread muscle cells, a marked increase in the expression level of dystrophin may be required for effective DMD gene therapy. Several studies have reported that the cellular free calcium is increased in dystrophic muscle and this increase activates calcium-dependent proteases, calpains. Because calpains degrade dystrophin, the silencing of calpain expression in dystrophic muscle cells may improve transgene expression of dystrophin. In this study, we used RNA interference (RNAi) to silence calpain expression in muscle cells. To this end, we constructed plasmid DNA expressing small interfering RNA (siRNA) targeting one of three isoforms of calpain, under the control of U6 promoter (psiCalp). Cationic liposome-mediated transfection of psiCalp targeting -calpain, m-calpain or p94 (calpain 3) resulted in significant reduction in the corresponding mRNA level in C2C12 myoblasts. The reduction in the mRNA level was associated with the reduced calpain activity in the cells. Total calpain activity in the gastrocnemius muscle of mdx mice (3- to 8-weeks-old) was found to be significantly greater than that of control (C57BL/10) mice. Intramuscular injection of 10100 g psiCalp into mdx muscle reduced the calpain activity. Then, plasmid DNA encoding a human micro-dystrophin cDNA (CS1, 4.9 kb), pCAG-CS1, was injected into the gastrocnemius muscle of 3-weeks-old mdx mice. Intramuscular injection of naked pCAG-CS1 resulted in significant, but low expression of the micro-dystrophin. On the other hand, co-injection of psiCalp greatly increased the expression of the micro-dystrophin, which was confirmed by immunostaining and immunoblot analysis. These results indicate that RNAi-mediated silencing of calpain expression is effective in increasing transgene expression of micro-dystrophin in mdx muscle. Although the silencing might be transient, the restoration of (micro-)dystrophin expression could inhibit aberrant activation of calpains in muscle cells.
 

20) Transplantation of SM/C-2.6+ Satellite Cells Transduced with Micro-Dystrophin CS1 cDNA by Lentiviral Vector into mdx Mice

Madoka Ikemoto, So-ichiro Fukada, Akiyoshi Uezumi, Satoru Masuda, Ampong Beryl Nyamekye, Hiroyuki Miyoshi, Hiroshi Yamamoto, Yuko Miyagoe-Suzuki, Shin'ichi Takeda Department of Molecular Therapy, National Institute of Neuroscience, NCNP, Kodaira, Tokyo, Japan; Subteam for Manipulation of Cell Fate, RIKEN Tsukuba Institute, Tsukuba, Ibaraki, Japan; Department of Immunology, Osaka University, Suita, Osaka, Japan

Duchenne muscular dystrophy (DMD) is an X-linked, lethal muscle disorder caused by mutations in the dystrophin gene (14 kb cDNA). Cell therapy is one of attractive approaches to the treatment of DMD. Satellite cells reside beneath the basal lamina and play major roles as stem cells in growth and repair of adult skeletal muscle. Satellite cells are expected to be an ideal cell source for cell therapy of DMD, but identification and accumulation of a pure satellite cell population in situ was very difficult. We developed a cell sorting system to purify quiescent satellite cells from intact mouse skeletal muscle using a recently developed novel monoclonal antibody SM/C-2.6 (Exp. Cell Res. 296: 245-255, 2004). First, to determine the regenerative capacity of SM/C-2.6+ cells, we injected three kinds of cells, freshly isolated quiescent cells, passaged cells and primary myoblasts derived from GFP-Tg mice into skeletal muscles of dystrophin-deficient mdx mice. Grafting of freshly isolated SM/C-2.6+ cells resulted in a large number of GFP-positive fibers at 4 weeks after the injection, whereas grafting of the same number of passaged SM/C-2.6+ cells and primary myoblasts lead to significantly fewer GFP-positive fibers. Surprisingly, culturing of SM/C-2.6+ cells only once after isolation hardly reduced their regenerative capacity. It is, therefore, possible to expand SM/C-2.6+ cells to some extent in vitro without reduction of their regenerative capacity. Next, to test whether autologous, genetically corrected satellite cells may represent a possible tool for the therapy of DMD, we tried to isolate SM/C-2.6+ cells from skeletal muscles of mdx mice and transduced them with lentiviral vector. In dystrophic muscle, the cells other than satellite cells exceedingly infiltrate and proliferate. Therefore, we purified satellite cells from mdx muscle using antibodies to additional surface markers (CD45, CD31 and Sca-1). When SM/C-2.6+, CD45-, CD31-, Sca-1- cells (mdx SM/C-2.6+ cells) were sorted and cultured in vitro for 4 days, more than 95% of them expressed MyoD. To genetically correct these cells, we produced a third-generation lentiviral vector expressing micro-dystrophin CS1 (BBRC. 293: 1265-1272, 2002; Mol Ther 10: 821-828, 2004) and then infected freshly isolated mdx SM/C-2.6+ cells with the lentiviral vector at MOI of 200 for 16 hours. Under these conditions, more than 90% of the cells were efficiently transduced. We, then, transplanted SM/C-2.6+ cells transduced with CS1 into mdx muscles. Four weeks after the injection, many fibers expressed micro-dystrophin on the sarcolemma. These data indicate that mdx SM/C-2.6+ cells transduced with micro-dystrophin by lentiviral vector can efficiently contribute to muscle regeneration. Thus, the combination of SM/C-2.6+ cells and lentiviral vector represents a promising approach to cell therapy of DMD.
 

21) Morpholino Antisense Oligonucleotide Induced Exon Skipping Efficiently Restores Dystrophin Expression in the mdx Mouse

Dominic J. Wells, Caroline McCulley, Anna Graham, Ke Liu, Kim E. Wells Cellular and Molecular Neuroscience, Imperial College London, London, United Kingdom

Although antisense oligonucleotides (AO) have been used most commonly to inhibit gene expression, they can also be used to modify splicing of the primary transcript by targeting the exon splicing enhancers or the 3'/5' splice sites. Recent studies have shown that the morpholino chemistry is particularly useful as such AOs are not readily degraded. The mdx mouse is dystrophin deficient due to a premature stop codon in exon 23. Using an AO that targets exon 23, we have examined the longevity of exon skipping by RT-PCR and dystrophin immunocytochemistry and western blots. Skipped dystrophin transcript can still be detected 14 weeks after a single intramuscular injection of morpholino AO and dystrophin is clearly present at least 4 months post treatment. This is substantially longer than the expression seen in similar studies using a 2-O-methyl phosphorothioate AO. We have used similar techniques to examine the dose/volume relationship in preparation for clinical trials of this approach in Duchenne muscular dystrophy (DMD). As the diaphragm of the mdx mouse is the muscle that most closely resembles the pathology in DMD, exhibiting substantial fibrosis and fibre loss, we have used the diaphragm to test the effect of increased fibrosis in reducing AO uptake by the muscle fibres by treating mice at different ages. Delivery of AO in mice younger than 2 weeks can largely prevent the development of dystrophic pathology and treatment at later ages reduces the membrane fragility as determined by uptake of Evans Blue dye. These pre-clinical studies demonstrate the effectiveness of the morpholino based AO in inducing long-lived expression of dystrophin and this approach is now being taken forward to a UK clinical trial in DMD by the MDEX Consortium.
 

22) Long-Term Expression of Mini-Human Dystrophin in Transgenic mdx Mice Improves Dystrophic Muscle Functions

Bing Wang, Juan Li, Chunlian Chen, Xiancheng Jiang, Terry O' Day, Jon Watchko, Xiao Xiao Orthopedic Surgery, University of Pittsburgh, Pittsburgh, PA; Pediatrics, Magee-Womens Research Institute, University of Pittsburgh, Pittsburgh, PA; Anatomy and Molecular Cell Biology, The State University of New York, New York, NY

BACKGROUND: Duchenne muscular dystrophy (DMD) is a lethal genetic muscle disorder caused by recessive mutations in the dystrophin gene and affects one in every 3500 males. The clinical and pathological characteristics of DMD patients show progressive myopathy of skeletal and cardiac muscles and premature death of patients. The human "mini- or micro -dystrophines" with different truncated central rod domains have been proven to ameliorate dystrophic pathology. In this study, our purpose is to investigate the long-term expression of human mini-dystrophin and therapeutic benefits in transgenic mdx mice.
METHODS: Four lines of transgenic mdx mouse (Tg-Dys3849-mdx) were generated. The 3.8 kb human mini-dystrophin containing five rods (R1-2, R22-24) of the central region and two hinges (H1 and H4) was driven by a truncated creatine-kinase promoter with two modified enhancers (dMCK). The gene expression cassette was cloned in an AAV vector plasmid for creation of transgenic mice. After five generations of back-crossing with the mdx mice, one of the lines containing a single copy of the minigene was used to test the gene expressions and muscle functions.
RESULTS: 1) the human mini-dystrophin expression was found a majority of the of skeletal muscles (Quad, GAS, TA, forelimb, abdominal, intercostals, thoracic and lumbar spinal muscles), but only very limited gene expressions in DIA and no expression in cardiac muscle; 2) Preference of gene expression was found in fast twitch myofibers over the slow fibers; 3) the DAGs such as sarcoglycans and nNOS were restored at sarcolemma and coincided with human mini dystrophin expression; 4) the morphology of dystrophic muscle expressing the human mini-dystorphin was improved and central nuclei were reduced to < 2%; 5) myofiber membrane integrity was improved by EBD test; 6) Improvement in treadmill running and grip force was observed in transgenic mice at 6 and 10 months when compared with the littermates (p<0.05); Also, tetanic force and specific force of TA muscle were significantly increased at 6, 10 and 20 months (p<0.05); 7) pseudohypertrophy was not found in GAS and TA mass (p<0.05).
Taken together, this study demonstrates that the human mini-dystrophin with 5 central rods can effectively ameliorate the pathology and improve the functions of the dystrophic muscles in mdx mice.
 

23) Rescue of Dystrophin in the GRMD Dog by Multi-Exon Skipping Using Engineered U7 snRNAs

Adeline Vulin, Aurelie Goyenvalle, Ines Barthelemy, France Leturcq, Jean-Claude Kaplan, Olivier Danos, Stephane Blot, Luis Garcia Maladie de Duchenne, Genethon - CNRS UMR 8115, Evry, France, Metropolitan; Laboratoire de Neurobiologie, ENVA, Maisons Alfort, France, Metropolitan; Laboratoire de Biochimie et de Genetique Mol, Institut Cochin, Paris, France, Metropolitan

Mutations in the dystrophin gene that create frame-shifts or premature stop codons on the mRNA are responsible of the severe Duchenne Muscular Dystrophy (DMD), while mutations that preserve the reading frame result in either milder or asymptomatic phenotypes. The exon skipping stratagem makes use of compounds designed to target key motifs on pre-mRNA to re-direct splicing in order to eliminate detrimental exons and rescue the production of shorter-proteins still functional. We have achieved persistent "on demand alternative splicing" that removes several consecutive exons on the dystrophin mRNA of the GRMD dog, by single administration of AAV vectors expressing antisense sequences coupled to a modified U7 short nuclear (sn) RNA. We report the sustained production of dystrophin at physiological levels in entire groups of muscles at the scale of a large size animal model of DMD.
 

24) Design and Optimization of U7snRNAs for Skipping of Exon 51 in DMD: Promising Tools for Future Clinical Trials

Aurelie Goyenvalle, Adeline Vulin, Stephanie Lorain, Annemieke Aartsma-Rus, Judith C. T. van Deutekom, Olivier Danos, Luis Garcia DMD Laboratory, Genethon, Evry, France, Metropolitan; Department of Human Genetics, Leiden University Medical Center, Leiden, Netherlands

Most cases of Duchenne muscular dystrophy (DMD) are caused by dystrophin gene mutations that disrupt the mRNA reading frame. In some cases, forced exclusion of a single exon can restore the reading frame, given rise to a shorter, but still functional dystrophin protein (so called quasi-dystrophin). One potential treatment of the disorder has utilized antisense oligoribonucleotide (AO) to induce removal of disease-associated exons during pre-mRNA processing. Indeed, this approach has been successfully used in DMD cells in vitro with antisense sequences against splice junctions of exon 51. Skipping of this exon would theoretically restore a functional quasi-dystrophin in a significant subset of DMD patients with 45-50,47-50,48-50, 49-50, 50 and 52 genotypes. However, since the AO are not self-renewed, they can not achieve long term correction. To overcome this limitation, we have introduced antisense sequences into small nuclear RNAs (snRNA) and vectorized them in AAV and lentiviral vectors.
We have designed AAV and lentiviral vectors harboring chimeric U7 snRNA carrying antisense sequences against exon 51 of the human dystrophin gene (U7-ex51). Lentiviral vectors expressing this U7-ex51 were tested on human myoblasts, whereas AAV vectors were injected in the transgenic hDMD mice (carrying the human dystrophin gene).
We confirmed the skipping of the exon 51 in vitro in human myoblasts after transduction with the lentiviral vector encoding U7-ex51 by RT-PCR. We also detected the skipping of the exon 51 after intramuscular injection of an AAV-U7ex51 vector in the transgenic hDMD mouse.
We have also tested the efficacy of these vectors to restore dystrophin expression in myoblasts from patients with 49-50 and 52 deletions.
In this study, we provide evidence that efficient skipping of exon 51 can be achieved in human cells and also in vivo after intramuscular injection in a transgenic hDMD mice through U7snRNA shuttle. These results offer very promising tools for clinical treatment of DMD.
 

25) Improvement of Muscle Mass Using shRNA Targeting Myostatin or Activin Receptor IIb

Julie Dumonceaux, Solenne Marie, Luis Garcia Maladie de Duchenne, Genethon, Evry, France

Duchenne Muscular Dystrophy (DMD) is the most severe degenerative disorder of skeletal and cardiac muscle. DMD patients show a progressive muscle weakness which begins in early childhood. Our goal is to stop this continuous muscle wasting.
Myostatin is a negative regulator of skeletal muscle mass. This protein binds to its cell-surface receptor (Activin receptor IIb, AcvRIIb) to inhibit both proliferation and differentiation of myoblasts. In vivo, it was shown that myostatin deficient mice show a double mass phenotype compared to wild type animals. Our hypothesis is that inactivation of myostatin function may consequently results in an increase of muscle mass in DMD patients as well as in its murine model, the mdx mouse.
We have investigated RNA interference technique to inhibit functions of myostatin. We decided to target AcvRIIb as well as the myostatin. Mice were injected intra muscularly using an AAV carrying the AcvRIIb shRNA or myostatin shRNA. One of the myostatin shRNA inhibits 95% of the mRNA expression. Preliminary results also show that a 50 % down regulation of AcvRIIb in mdx mice results in an 15% increase in muscle mass. The next step is now to combine on a same AAV vector the exon skipping strategy (ie AAV-U7-SD23/BP22) and the inactivation of myostatin function (ie AAV-AcvRIIb shRNA). This vector may serve to both rescue dystrophin and improve muscle mass.
 

26) Haute Couture Vectors: Solving Clinical Problems with Custom Designed AAV Vectors

Dawn E. Bowles, Chengwen Li, Paul E. Monahan, Joseph E. Rabinowitz, Mavis McKenna, Jude Samulski Gene Therapy Center, University of North Carolina at Chapel Hill, Chapel Hill, NC; Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, FL

Compelling evidence that therapeutic transgene levels can be achieved from adeno-associated virus (AAV) vectors has not been observed in any human gene therapy application to date. We show that AAV capsids can be tailored to solve such clinical problems and describe here a new generation of AAV vectors approved for use in clinical studies.
The availability of capsid DNA sequences from AAV serotypes exhibiting elevated tissue transduction profiles, the absence of cross-neutralizing antibody between certain serotypes (e.g. AAV2 and AAV1), and the three-dimensional structure of the AAV2 capsid served as a roadmap for AAV2 capsid surface positions to change as well as replacement amino acid candidates in the generation of these new vectors.
A subset of these rationally designed AAV2 capsid variants exhibited enhanced skeletal muscle transduction, diminished recognition by human sera, and the ability to overcome the presence of AAV2 neutralizing antibodies all without disrupting current AAV2 purification parameters. These chimeric AAV vector displayed identical viral yield as parent virus AAV2, vector stability, and long-term gene expression in vivo. Further engineering determined that the insertion of only one amino acid in the AAV2 capsid was responsible for both the phenotypes of skeletal muscle enhanced transduction as well as resistance to anti-AAV2 neutralizing antibodies. Validation of these chimeric AAV vectors in clinical trials is on-going and outcome should provide essential data required for testing in human disorders such as muscular dystrophy and hemophilia B.
Notes: DEB and CL contributed equally to this work. Present addresses of DEB and JER: Department of Surgery, Duke University, DUMC, Durham, NC (DEB). Center for Translational Medicine, Thomas Jefferson University, Philadelphia, PA (JER).
 

27) Physiological Correction of Glycogen Storage Disease Type II Using Adeno-Associated Virus Serotype 1 Vectors

Cathryn Mah, Christina A. Pacak, Kerry O. Cresawn, Lara R. DeRuisseau, Sean Germain, Melissa A. Lewis, David D. Fuller, Barry J. Byrne Pediatrics, Div. Cell and Molecular Therapy and Pediatric Cardiology, University of Florida, Gainesville, FL; Powell Gene Therapy Center, University of Florida, Gainesville, FL; Interdisciplinary Program in Biomedical Sciences, Genetics Advanced Concentration, University of Florida, Gainesville, FL; Graduate Program in the Dept of Physiological Sciences, College of Veterinary Medicine, University of Florida, Gainesville, FL; Physical Therapy, College of Public Health and Health Professions, University of Florida, Gainesville, FL

Glycogen storage disease type II (GSDII) is caused by a lack of functional lysosomal acid -glucosidase (GAA) and results in massive storage of glycogen in lysosomal compartments, ultimately leading to fatal severe hypertrophic cardiomyopathy and respiratory insufficiency. Previously, we demonstrated the ability of a single intravenous administration of recombinant adeno-associated virus serotype 1 (rAAV2/1) vector to restore therapeutic levels of cardiac and diaphragmatic GAA enzymatic activity with concomitant clearance of glycogen in vivo in a mouse model of GSDII (Gaa^-/-). We have further characterized both cardiac and respiratory function in rAAV2/1-treated animals one year post-treatment. Similar to the GSDII patient population, electrocardiogram (ECG) measurements (P-R interval) are significantly shortened in the mouse model. In rAAV2/1-treated mice, we show a significant improvement in cardiac conductance with prolonged P-R intervals of 39.34+1.6 ms, as compared to untreated controls (35.58+0.57 ms) (p<0.05). In addition, using cardiac magnetic resonance imaging (MRI) we note a marked decrease in cardiac left ventricular mass from 181.99+10.70 mg in untreated age-matched controls to 141.97+19.15 mg in the rAAV2/1-treated mice. Furthermore the mice displayed increased diaphragmatic contractile force to approximately 90% of wild-type peak forces with corresponding significantly improved ventilation (particularly in frequency, minute ventilation, and peak inspiratory flow), as measured using barometric whole body plethysmography. These results demonstrate that in addition to biochemical and histological correction, rAAV2/1 vectors can mediate sustained physiological correction of both cardiac and respiratory function in a model of fatal cardiomyopathy and muscular dystrophy.
 

28) Optimizing Intron Splicing Signal Overcomes the mRNA Accumulation Barrier in Trans-Splicing AAV Vectors

Yi Lai, Dongsheng Duan Department of Molecular Microbiology and Immunology, School of Medicine, University of Missouri-Columbia, Columbia, MO

Trans-splicing adeno-associated viral (AAV) vectors hold great promise in many gene therapy applications. We have recently shown that rational selection of the gene splitting site and the endogenous intron sequence can lead to extremely efficient trans-splicing vectors compatible to that of a single intact AAV vector (Lai et al Nature Biotechnology 23:1435, 2005). A key factor in constructing effective trans-splicing vectors is the mRNA production from the reconstituted viral genome. In the dystrophin gene (a therapeutic gene for Duchenne muscular dystrophy), the 60/60/61 (exon/intron/exon) junction yields the highest mRNA level while the 63/63/64 junction is the most efficiently spliced junction when tested in the reconstituted trans-splicing viral genome. However, only vectors based on the 60/60/61 junction can produce therapeutic level protein. Many endogenous introns carry sub-optimal splicing signals. It is not clear whether optimizing intron sequence can further improve trans-splicing AAV vectors. In this study, we evaluated the effect of replacing the endogenous intron with a synthetic intron that matches perfectly with the conserved motif. We first generated two additional constructs p60/synthetic/61 and p63/synthetic/64. In these constructs, the endogenous introns were replaced by the synthetic intron in p60/60/61 and p63/63/64, respectively. Similar to the parent constructs, the newly synthesized constructs also carried the double-D inverted terminal repeat junction in the middle of the intron. RNase protection assay (RPA) was used to determine the level of unspliced and spliced RNA, and the relative splicing efficiency. At 48 hrs after transfection, RNA was extracted for RPA. The splicing indices in intron 60 are quite compatible to those of the conserved motif. Substitution of the endogenous intron with the synthetic intron resulted in marginal but statistically not significant improvement. The mRNA level in p60/60/61 was 236.5 + 132.5 (relative unit) and it was 300.2 + 86.6 in p60/synthetic/61 (p > 0.05). However, the same replacement in the 63/64 exon junction led to a three-fold increase in accumulated mRNA from 124.3 + 5.8 to 362.4 + 32.3 (p < 0.05). We next examined the relative splicing efficiency. Interestingly, the synthetic intron did not augment the already efficient splicing in the 63/63/64 junction. However, the relatively poor splicing in p60/60/61 was improved. The ratio of spliced to unspliced RNA increased from 2.8 + 0.3 in p60/60/61 to 7.7 + 1.9 in p60/synthetic/61. The percentage of splicing also increased from 72.6 % to 85.2 %.To further explore the molecular mechanisms, we quantified the pre-mRNA stability. At 48 hrs after transfection, actinomycin D was added to cell culture to stop de novo RNA synthesis. RNA was extracted at 0, 0.5, 1 and 3 hrs after actinomycin D treatment. In these studies, we observed increased pre-mRNA stability in both 60/synthetic/61 and 63/synthetic/64 transcripts. Taken together, our results suggest that optimizing intron sequence may boost the transduction efficiency of trans-splicing AAV vectors.
 

29) Efficient Dystrophin Production in Response to Local and Systemic Administration of Morpholino Antisense Oligonucleotides in the Mdx Mouse

Ian R. Graham, Francesca C. Thorogood, Helen Foster, George Dickson Centre for Biomedical Sciences, Royal Holloway - University of London, Egham, Surrey, United Kingdom

Duchenne muscular dystrophy (DMD) is an X-linked recessive inherited disease that affects 1 in 3,500 male births. It is characterised by severe muscle wasting and muscle degeneration. Affected patients are frequently wheelchair-bound prior to 12 years and usually die in their late teens or early twenties as a result of respiratory or cardiac failure. DMD arises as a result of frameshift mutations (often gross deletions) in the gene encoding dystrophin, a 427kDa protein of the muscle sarcolemma that functions as a molecular scaffold connecting the intracellular actin cytoskeleton with the extracellular matrix. Such mutations result in a complete absence of dystrophin protein and lead to the DMD phenotype, for which there is currently no cure.
Gene therapy approaches have traditionally been based on gene augmentation strategies, in which a functional copy of the dystrophin cDNA, or truncations thereof, has been introduced on viral or plasmid vectors. In an alternative strategy, we and others have been developing the use of antisense oligonucleotides (AO) to induce the exclusion of specific exons during the splicing of dystrophin pre-mRNA. We have used the mdx mouse, a naturally-occurring model of DMD carrying a stop codon in exon 23 of the dystrophin gene, to demonstrate that exclusion (skipping) of exon 23 in response to AO does indeed result in de novo production of dystrophin by modulation of the splicing pathway.
Recent advances in oligonucleotide chemistry and design have allowed us to compare the efficiency of delivery and bioactivity of AOs based on morpholino phosphorodiamidate backbones with those of the previous 2'-O-methyl chemistry. Again using the mdx mouse, we have performed dose-response and time-course studies of morpholino AOs targeted to exon 23, by both intramuscular and intravenous delivery. In this way, we have demonstrated widespread and efficient AO-induced dystrophin production by both delivery routes, in a manner which shows AO therapy to be a potentially viable treatment for DMD in a clinical setting.
 

30) Effective Repetitive Dystrophin Gene Transfer into Skeletal Muscle of Adult mdx Mice Using a Helper-Dependent Adenovirus Vector Expressing the Coxsackievirus and Adenovirus Receptor (CAR) and Dystrophin

Yuji Uchida, Yasushi Maeda, En Kimura, Makoto Uchino Department of Neurology, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan

The helper-dependent adenovirus (HDAd) vector is less immunogenic and has a larger cloning capacity of up to 37 kb enough to carry the full-length dystrophin cDNA. However, high and long-term expression of dystrophin transduced to mature muscle still remains difficult. One of the main reasons for this is that the expression of the coxsakievirus and adenovirus receptor (CAR) is very low in mature muscle. We have constructed two different HDAd vectors. One contains the LacZ and the murine full-length dystrophin expression cassette (HDAdLacZ-dys), and the other is a new, improved vector containing the CAR and the dystrophin expression cassette (HDAdCAR-dys). We initially demonstrated high dystrophin expression and prevention of the dystrophic pathology in mdx muscle injected during the neonatal phase with HDAdLacZ-dys. Furthermore, we demonstrated that repeated injections of HDAdCAR-dys into mature muscle led to approximately nine times greater dystrophin-positive fibers in number than a single injection, thereby recovering the expression of dystrophin-associated proteins. This data has also shown that HDAdCAR-dys enabled administration of adenovirus (Ad) vector to the host with pre-existing immunity to the same serotype of Ad. Repetitive injections of the HDAd vector containing the CAR and the dystrophin expression cassette could improve the efficiency of subsequent dystrophin gene transfer to mature mdx muscle. This result suggests that our new HDAd vector will provide a novel gene therapy strategy for Duchenne muscular dystrophy. In addition, we injected mdx mice intraperitoneally with HDAdLacZ-dys to transduce into diaphragm.