ABSTRACTS THAT WILL BE PRESENTED IN THE 7TH ANNUAL MEETING OF THE AMERICAN SOCIETY OF GENE THERAPY, JUNE 2-6, MINNEAPOLIS, MINNESOTA

1) Micro-Utrophin as a Therapeutic Protein in rAAV Mediated Gene Therapy for Duchenne Muscular Dystrophy

Michael J. Blankinship, Paul Gregorevic, Jeffrey S. Chamberlain Neurology, University of Washington School of Medicine, Seattle, WA

Vectors based on recombinant adeno-associated virus (rAAV) have garnered significant interest in gene replacement therapies for Duchenne muscular dystrophy. Several serotypes of rAAV have been shown to transduce skeletal muscle with high efficiency and low toxicity. These vectors suggest a possible therapeutic approach where dystrophin expression cassettes could be delivered to the striated musculature of an affected individual. However, a significant constraint inherent in rAAV vectors is their relatively small packaging capacity of approximately 5 kb. This packaging capacity is of great concern in attempting to deliver dystrophin via rAAV as the dystrophin cDNA is approximately 14 kb. This limitation has been overcome by engineering dystrophin proteins encompassing large deletions, which nonetheless remain highly functional. An additional concern in gene replacement therapies is the possibility of a patient’s immune system viewing the therapeutic protein as a foreign antigen and raising a detrimental immune response, as the patient may not normally express the therapeutic protein. In Duchenne muscular dystrophy, a possible alternative to dystrophin replacement is utrophin over-expression. Utrophin is an autosommal homologue of dystrophin that is expressed in patients and should be viewed as a self antigen. Utrophin over-expression has been shown to functionally rescue a dystrophin deficient mouse model of muscular dystrophy, though the utrophin cDNA is also too large to be packaged into rAAV vectors without modification. Here we report and characterize a micro-utrophin protein based precisely on our previously reported, highly functional micro-dystrophin ( ΔR4-R23/ ΔCT). Despite high, uniform expression and proper localization to the sarcolemma, this protein is not as efficient as micro-dystrophin in rescuing the dystrophic phenotype. Significant pathology is seen on a gross histological level despite restoration of DGC proteins to the sarcolemma. These findings highlight potential difficulties in micro-utrophin based therapies for Duchenne muscular dystrophy via rAAV, and suggest that domain engineering may be required to enable generation of a functional micro-utrophin/dystrophin hybrid

2)Gender Differences in Transplantation Efficiency Using Muscle-Derived Stem Cells for Muscular Dystrophy

Bridget M. Deasy, Aiping Lu, Burhan M. Gharaibeh, Michele Jones, Jessica Tebbets, Johhny Huard Bioengineering, University of Pittsburgh, Pittsburgh, PA; Growth and Development Laboratory, Children's Hospital of Pittsburgh, Pittsburgh, PA; Molecular Genetics & Biochemistry and Orthopedic Surgery, University of Pittsburgh Medical Center, Pittsburgh, PA

Duchenne muscular dystrophy (DMD) is a devastating X-linked muscle disease characterized by progressive muscle weakness due to the lack of dystrophin expression at the sarcolemma of muscle fibers. Transplantation of normal myoblasts into diseased muscle provides donor myoblasts that fuse with dystrophic muscle fibers and restore dystrophin. This process enables transient dystrophin delivery and improved strength in the injected dystrophic muscle. However, the approach has limitations, including immune rejection, poor cellular survival rates, and limited dissemination of the donor cells. The outcome of this cell transplantation therapy has been improved in the murine DMD model (mdx) by using muscle-derived stem cells (MDSCs). This enhanced success appears to be attributable to several unique features of stem cells: 1) self-renewal with production of identical progeny, 2) appearance early in development and persistence throughout life, and 3) long-term proliferation and multipotency.
The purpose of this study was to explore potential gender differences on transplantation efficiency. Specifically, we sought to determine whether there is a difference in 1) the regeneration efficiency of male- versus female-derived MDSCs and 2) the receptiveness of the male and female mdx hosts to transplantation.
Our studies found that a donor population of MT (male, three-weeks donor) did not have the same level of engraftment as FT (female, three-weeks) in terms of dystrophin delivery to mdx animals. Female MDSCs are more efficient than male MDSCs in facilitating dystrophin delivery and muscle regeneration in the murine muscular dystrophy model While we recognize there is large variability among engraftments, we found overall that the average regeneration index for the female populations was 516 dystrophin positive myofibers per 10⁵ donor cells, and 135 fibers/10⁵ cells for male populations (p<0.05). Several in vitro characteristics were explored to understand the in vivo differences. While both populations were isolated by the preplate technique and were capable of extended replicative lifetime, there were differences in proliferation rate, desmin expression and CD34 expression which may help to explain the differences in regeneration efficiency.
Examination of the host receptiveness also revealed that female MDSC were able to achieve a much larger engraftments in female mdx hosts as compared to their performance in male mdx hosts. High engraftment was observed for female cells into female host (RI= 556 fibers/10^5 cells), which supported previous results. However, these same cells have a lower engraftment when injected to age-matched male hosts (RI= 276 fibers/10^5 cells) (p=.064).Our results imply that gender-related differences play a role in the transplantation efficiency of MDSCs. We are conducting an ongoing analysis in an attempt to better understand the mechanism by which female MDSCs support higher engraftment than male MDSCs. In addition, we are exploring possible immune responses that the male host may have to female donor cells.

3) Therapeutic Antisense-Induced Exon Skipping for Duchenne Muscular Dystrophy

Annemieke Aartsma-Rus, Anneke A. M. Janson, Wendy E. Kaman, Mattie Bremmer-Bout, Christa L. de Winter, Habte F. Teshale, Gert-Jan B. van Ommen, Johan T. den Dunnen, Judith C. T. van Deutekom Human Genetics, Leiden University Medical Center, Leiden, Netherlands

The severe Duchenne muscular dystrophy (DMD) is mostly caused by frame disrupting mutations in the dystrophin gene, which result in non-functional dystrophin proteins. Mutations that keep the reading frame intact give rise to internally deleted, semi-functional dystrophins and result in the milder Becker muscular dystrophy (BMD). Antisense oligonucleotides (AONs) have the potential to modulate the pre-mRNA splicing such that a specific exon is skipped. As a result, the reading frame can be restored, which allows the synthesis of a BMD-like dystrophin.
To date, we have induced the skipping of 20 different DMD exons in human control myotubes and confirmed the therapeutic applicability of the strategy in myotube cultures from 10 different DMD patients. Following transfection of specific AONs dystrophin synthesis was restored in over 75% of treated myotubes. Furthermore, we recently demonstrated the feasibility of skipping two and even multiple consecutive exons, simultaneously. This double- and multi-exon skipping would not only further increase the therapeutic applicability to over 90% of patients, but also render this therapy significantly less mutation-specific.
For future clinical applications the optimal AON induces high levels of exon skipping at low levels of cytotoxicity. Thus far we have used AONs containing 2'-O-methyl RNA with a full-length phosphorothioate backbone (2OMePS). We have compared the efficacy and efficiency of our most efficient exon 46 2OMePS AON to those of a morpholino, a locked nucleic acid (LNA) and a peptide nucleic acid (PNA) AON. The LNA induced higher levels of exon skipping than 2OMePS in patient (>98% vs. 85%) and control (>85% vs. 20%) myotube cultures. The morpholino only induced low levels of exon skipping both in patient and control (~5%), while the PNA was ineffective. We then compared the sequence specificity of the 2OMePS and LNA AONs; one mismatch resulted in an over 2-fold decrease of activity for the 2OMePS AONs, while LNA AONs containing one or two mismatches were nearly as effective as the wild type LNA. Based on these results we concluded that 2OMePS are currently the most favorable compounds.
Finally, we have previously engineered a mouse model that contains the entire human DMD gene (2.6 Mb) integrated into the murine genome (hDMD mouse). These transgenic mice uniquely allow for the preclinical testing of human-specific AONs in vivo. In fact, we have injected AONs targeting human exons 44, 46 and 49 into the m. gastrocnemicus of hDMD mice, and showed that the skipping of the human exons (but not the murine exons) was indeed specifically induced. Furthermore, a time course experiment revealed that the exon skipping effect could be observed as early as 1 day post-injection and was persistent for at least 28 days. Although the results to date appear to support short-term therapeutic promise of AONs, parameters such as choice of target sequence, oligochemistry, and methodology for safe and efficient delivery will have to be further optimized.

4) New Canine Models of Duchenne Muscular Dystrophy: Identification and Molecular Characterization

Bruce F. Smith, Lucia Alvarez, Kerriann Sparks Scott-Ritchey Research Center, Auburn University, Auburn, AL

Duchenne Muscular Dystrophy (DMD) presents a series of significant challenges to the development of gene therapy approaches, including the frequency of new mutations, the size of the gene and mRNA and the complexity of the mutations involved. Animal models can significantly accelerate the process for development of novel therapies if they accurately mimic the human disease. To date, the only animal that develops disease with a course and severity similar to humans, without requiring additional mutations or manipulations, is the dog. Several canine models have been identified and their mutations characterized, confirming that this disease occurs at a relatively high frequency, can have variable effects, and is the result of many different mutations. Unlike other canine inherited diseases, Duchenne-like Muscular Dystrophy occurs spontaneously in multiple families within in a breed, leading to more than one mutation in a given breed of dog. Using a rapid PCR based screen, we have identified the putative mutations in two canine models of DMD. Data from a Labrador Retriever family and a Welsh Corgi family indicate that the mutations in both families consist of the precise insertion of repetitive DNA elements in the mRNA between exon pairs. The mutations involve different repetitive sequences and different exon pairs in each family. In addition, affected animals from another distinct Labrador Retriever family and a West Highland White family have been identified. Preliminary data supporting the independent nature of these mutations as well as their localization within the gene will be presented. The relatively large size, intact immune system, and potential to measure beneficial effects combined with the assortment of different mutations available provide an excellent resource in which gene therapy approaches for DMD can be tested.

5) A Novel MiniDys-eGFP Fusion Gene for Developing Cell-Based Therapies of Duchenne Muscular Dystrophy

Sheng Li, En Kimura, Leonard Meuse, Xin Ye, Brent Fall, Steven D. Hauschka, John Faulkner, Jeffrey S. Chamberlain Department of Neurology, University of Washington School of Medicine, Seattle, WA; Department of Medicine, University of Washington School of Medicine, Seattle, WA; Department of Biochemistry, University of Washington School of Medicine, Seattle, WA; Department of Physiology and Biomedical Engineering, University of Michigan, Ann Arbor, MI

Duchenne muscular dystrophy (DMD) results from mutations in the largest known gene, dystrophin. Gene therapy for DMD will require methods to systemically deliver a therapeutic dystrophin gene to widely distributed muscles. Recent observations indicating that vascularly transplanted adult stem cells can home and become muscle cells in an appropriate micro-environment has suggested one possible method for delivering genes to muscle. A smaller, easily traceable, and functional dystrophin would facilitate experiments aimed at improving ex vivo cell-based strategies. We previously found that large portions of the dystrophin central rod domain (Hinge 2 to Repeat 19) and the C-terminal domain (encoded on exons 71 to Exon 78) are not essential for the full function of dystrophin, as assayed by the ability to prevent and partially reverse morphological and fucnional abnormalities in mdx mouse skeletal muscles. Here, we engineered a 5.7-kb MiniDys-eGFP fusion gene by replacing the C-terminal domain of mini-dystrophin (DH2-R19) with an enhanced green fluorescence gene coding sequence and by trimming down the size of 5’ and 3’ UTR of dystrophin. This fusion gene can be inserted into lentiviral vectors and efficiently delivered into a variety of cell types in vitro. This MiniDys-eGFP fusion protein was found to fully prevent dystrophy of multiple skeletal muscles in transgenic mdx mice carrying the fusion gene under the control of the human a-skeletal actin (HSA) gene promoter. This green fusion protein was easily observed by fluorescence microscopy on the sarcolemma of skeletal muscle fibers of the transgenic mdx mice. Furthermore, donor-derived GFP-positive myofibers were detected in mdx recipient muscles transplanted with either whole bone marrow cells or primary myoblasts isolated from the MiniDys-eGFP transgenic mice. These data indicate that the smaller, easily traceable, and functional MiniDys-eGFP will be useful for developing ex vivo cell-based gene therapies for DMD.
 
 6) Expressing Full-Length Dystrophin in 50% Cardiomyocytes Corrects Cardiomyopathy in the Mdx Mouse Model for Duchenne Muscular Dystrophy

Yongping Yue, Jeffrey W. Skimming, Mingju Liu, Yujiang Fang, Tammy Strawn, Dongsheng Duan Molecular Microbiology and Immunology, University of Missouri, Columbia, MO; Child Health, Medical Pharmacology and Physiology, University of Missouri, Columbia, MO

Cardiomyopathy is a major determinant of the clinical outcome in Duchenne and Becker muscular dystrophy (DMD, BMD). Nearly every DMD and BMD patient suffers from some degree of cardiomyopathy. More then one tenth of DMD patients eventually die of heart failure. Clinical success of DMD gene therapy will depend upon functional improvement in both skeletal and cardiac muscle. Substantial progress has been made in DMD skeletal muscle disease gene therapy. However, few studies have been done in DMD cardiomyopathy gene therapy. We recently reported that micro-dystrophin was equally efficient in restoring the dystrophin-glycoprotein complex and maintaining sarcolemma integrity in the mdx heart (Yue et al Circulation 108:1626,2003). The minimal number of dystrophin expressing cells needed for cardiomyopathy therapy has not been determined however. In this study, we used female heterozygous mice (F1 from BL10 and mdx crossing) as an experimental model to evaluate whether dystrophin expression in half of the cardiomyocytes was enough to improve heart function in mdx mice. Consistent with the random X-chromosome inactivation theory, we found that 51.22% and 55.40% of the heart cells were expression dystrophin in maternal and paternal heterozygous mice respectively. The mdx mouse hearts were heavier than the BL10 hearts. Interestingly, weights of the heterozygous mice hearts were similar to those of the BL10. In contrast to previous reports of the benign histology in the mdx hearts, we detected fibrosis in 85.71% of the mdx hearts (N=42). More than half of the fibrosis was in the range of medium-to-large size. Only 43.59% of the heterozygous mice had hearts that contained fibrous regions, and the majority of the fibrosis was localized to small areas. To determine whether full-length dystrophin expression in half of the cardiomyocytes can protect the heart from mechanical-stress induced injury, we challenged the hearts with the inotrope b-isoproterenol. After administrating a vital dye, Evans blue (EBD), we found that 11.26 ± 3.40 % of the heart area was EBD positive in mdx mice. In the heterozygous mouse hearts, the EBD positive area was reduced to 2.37 ± 0.70 %. This result suggests that a significant improvement in cardiomyocyte sarcolemma integrity has been achieved in the heterozygous mouse hearts. In summary, our results suggest that a 50% correction in the mdx heart is sufficient to ameliorate cardiomyopathy in mdx mice.

7) Expression of Normal Dystrophin Following Myoblast Transplantation to Duchenne Muscular Dystrophy Patients

Jacques P. Tremblay, Daniel Skuk, Bouchard Jean-Pierre, Michel Sylvain, Roy Raynald, Goulet Marilyne, Roy Brigitte, Pierre Chapdelaine, Dugré Francine, Jean-Guy Lachance, Louise Deschènes, Hélène Senay Centre de Recherche du CHUQ, Centre Hospitalier Universitaire de Québec, Québec, QC, Canada; Neurologie, Centre Hospitalier Affilier de Québec, Québec, QC, Canada

Three Duchenne muscular dystrophy (DMD) patients received injections of myogenic cells obtained from skeletal muscle biopsies of normal donors. Cells were injected in 1 cm³ of the Tibialis anterior by 25 parallel injections. We performed similar patterns of saline injections in the contralateral muscles as controls. The patients received tacrolimus for immunosuppression. Muscle biopsies were performed at the injected sites 4 weeks later. We observed dystrophin-positive myofibers in the cell-grafted sites: 9 % (patient 1), 6.8 % (patient 2) and 11 % (patient 3). Since patients 1 and 2 had identified dystrophin-gene deletions these results were obtained using mAbs specifically to epitopes coded by the deleted exons. Donor-dystrophin was absent in the control sites. Patient 3 had exon duplication and thus specific donor-dystrophin detection was not possible. However there was 4-fold more dystrophin-positive myofibers in the cell-grafted than in the control site. Donor-dystrophin transcripts were detected by RT-PCR (using primers reacting with a sequence in the deleted exons) only in the cell-grafted sites in patients 1 and 2. Dystrophin transcripts were more abundant in the cell-grafted than in the control site in patient 3. Therefore, significant dystrophin expression can be obtained in the skeletal muscles of DMD patients following specific conditions of cell delivery and immunosuppression.

8) rAAV-Mediated Gene Therapy To Treat Limb Girdle Muscular Dystrophy Type 2D (LGMD-2D)

Christina A. Pacak, Denise Cloutier, Irene Zolotukhin, Gabriel S. Gaidosh, Kevin Campbell, Glenn A. Walter, Barry J. Byrne Molecular Genetics and Microbiology, Powell Gene Therapy Center, University of Florida, Gainesville, FL; Howard Hughes Medical Institute, Physiology and Physics, University of Iowa, Iowa City, IA

The long-term goal of this project is to develop a clinically relevant gene therapy approach for the treatment of limb girdle muscular dystrophy type 2D (LGMD-2D). LGMD-2D is the result of mutations in the alpha sarcoglycan (ASG) gene and is characterized by the progressive development of lesions in skeletal muscle due to deterioration of the sarcolemma. Our initial objective has been to demonstrate that recombinant adeno-associated virus (rAAV) can be used as a vehicle for delivery of the ASG gene to dystrophic muscle. We first undertook an in vitro promoter comparison study in which we determined that AAV serotype 1 with both the CMV and a modified form of the murine creatine kinase promoter that confers muscle specific expression, tMCK, have the ability to drive ASG gene expression in differentiated ASG-/- myoblasts at nearly equal levels. To evaluate gene delivery in vivo we have developed a non-invasive imaging assay using MRI that enables us to locate and measure the random development of lesions within skeletal muscle of ASG-/- mice. 1 and 2-month-old ASG -/- mice were imaged to determine the initial size and location of lesions in muscles of the lower extremities. Less than 15% of the tibialis anterior (TA) muscles from mice imaged at 1 month and approximately 90% of TA muscles imaged at 2 months showed lesion development. Therefore, we have established that there is a critical window for prevention and/or correction of lesion development in specific muscles during the first few weeks of life in the ASG-/- mouse. Both age groups were injected in one TA with 1 x 10¹¹ viral particles of either rAAV1-tMCK-ASG or rAAV2-CMV-ASG. For 4 months , monthly MRI was performed to observe lesion development. These images have enabled us to generate T2 maps to quantify areas of elevated intensity in specific regions of interest. Upon sacrifice, we have performed force mechanics to evaluate the ability of our therapy to provide not only physiological but also functional correction to dystrophic muscle. We have used immunohistochemistry to determine ASG expression and Evans Blue Dye (EBD) to elucidate the presence of lesions on tissue sections and thereby further confirm the MRI data. Tri-chrome staining was performed to identify lesions that have become infiltrated with collagen. Initial MRI, immunohistochemistry and force mechanics data indicate that each vector is able to confer both physiological and functional correction in the ASG-/- mouse model however, in both cases; the majority of transgene expression was detected along the needle path of the injection site. In a related experiment, <12 hour old ASG-/- pup legs have been injected with 1 x 10¹¹ viral particles of either AAV1-tMCK-ASG or AAV1-tMCK-LacZ to determine if better vector distribution (and ultimately more successful correction) can be obtained when therapy is delivered at an earlier point in development. In conclusion, our preliminary data suggests that (rAAV) is an effective vehicle for delivery of the ASG gene in ASG-/- skeletal muscle and is therefore a promising vector for gene delivery in vivo but time of injection and vector distribution may be critical factors in successful treatment.
Keywords: Musculo-Skeletal Diseases; Viral Gene Transfer; Targeted Gene Expression

 9) Transgenic Expression of Dp116 in Muscle Does Not Ameliorate Dystrophy in mdx^4cv Mice

Luke M. Judge, Jeffrey S. Chamberlain Molecular and Cellular Biology, Medical Scientist Training Programs, University of Washington, Seattle, WA; Neurology and Biochemistry, University of Washington, Seattle, WA

Duchenne Muscular Dystrophy (DMD) is caused by absence of the protein dystrophin in skeletal muscle. Dystrophin completes a link between the extracellular matrix and the cytoskeleton by binding to β-dystroglycan and actin. It is also required for stable expression of the dystrophin-glycoprotein complex (DGC) that is thought to be involved in cell signaling. Thus, dystrophin may play both mechanical and signaling roles in muscle fibers. The mechanical defect in DMD is likely to be the initial cause of injury to the sarcolemma, which is then exacerbated by altered cellular signaling that results in increased apoptosis, inflammation, and fibrosis. Correction of the signaling defects could potentially reduce the severity and progression of muscle damage. In order to evaluate the relative importance of the signaling and mechanical functions of dystrophin we generated transgenic mice that express D116, the peripheral nerve-specific isoform of dystrophin, in skeletal muscle. Dp116 contains the complete WW, cysteine-rich and C-terminal domains of dystrophin, which are necessary for stabilization of the DGC and associated molecules important for cell signaling. However, Dp116 retains only two complete spectrin-like repeats from the central rod domain and does not contain any known actin-binding domains, thus it should not contribute to mechanical stabilization of the sarcolemma. Dp116 transgenic mice were backcrossed onto the mdx^4cv strain, a model of DMD. Despite expression of Dp116 at the sarcolemma these mice have a dystrophic phenotype that appears to be at least as severe as that of mdx alone. This result implies that portions of the N-terminal and rod domains of dystrophin are absolutely required for prevention of the dystrophic pathology

10) Systemic Gene Transfer to Striated Muscles Using rAAV6 Vectors

Paul Gregorevic, Michael J. Blankinship, James M. Allen, Leonard Meuse, Jay Han, Suzanne Oakley, Jeffrey S. Chamberlain Muscular Dystrophy Co-Operative Research Center, Department of Neurology, University of Washington, Seattle, WA

Human mortality is severely affected by diseases of the cardiac and skeletal musculature. Genetic interventions are being developed for these diseases, but are limited by an inability to achieve widespread gene transfer to the heart and numerous skeletal muscles of an adult mammal. We have observed that recombinant adeno-associated viral vectors (rAAV vectors) comprising serotype type 6 capsid proteins potently transduce striated muscles following direct intramuscular injection. In subsequent experiments we have identified procedures for administering rAAV6 vectors intravascularly to conscious adult mice that achieve high-level transgene expression (up to 8,000 fold increases in indices over control values) in the vast majority of cardiac and skeletal muscle fibers. These data are the first to demonstrate that extensive transduction of both the cardiac and skeletal musculature is achievable in an adult mammal using a single, minimally invasive intravenous injection of rAAV6 vectors. We have observed that transgene expression in striated muscle fibers following vector administration is influenced by the design of the expression cassette delivered and present data demonstrating that muscle-restricted transgene expression reduces the incidence of immunological reaction against the transgene product compared with a constitutively-driven expression cassette. To assess the therapeutic potential of these novel gene delivery techniques, we have begun administering rAAV6 vectors containing therapeutic expression cassettes to murine models of Duchenne muscular dystrophy. Treated dystrophic mdx mice exhibit widespread expression of therapeutic dystrophin-based proteins throughout the striated musculature in a manner that is sufficient to reduce pathological features of the dystrophic phenotype, including susceptibility to contraction induced injury, and serum creatine kinase (an indicator of muscle degeneration). Having established a method of facilitating widespread gene delivery throughout the muscles of adult mice, we are now evaluating modifications of these techniques for increased gene delivery efficiency. Here we summarize our understanding of the factors influencing rAAV-mediated systemic gene delivery to striated muscles, and the therapeutic potential of such techniques for the treatment of muscular dystrophies.

11) Lentivirus Mediated Dystrophin Expression in mdx Muscles

En Kimura, Sheng Li, Brent Fall, Sanna Sawatzki, Leonard Meuse, Jeffery S. Chamberlain Department of Neurology, University of Washington School of Medicine, Seattle, WA

To study candidate treatments for Duchenne muscular dystrophy, we have generated a series of lentiviral vectors that express various reporter genes and mini-dystrophin cassettes and tested their ability to transduce a variety of cell types in vitro and in vivo . Direct injection of lentiviral vectors into adult skeletal muscle resulted in significantly lower levels of gene expression than were obtained using either AAV or adenoviral vectors.
Since an advantage of lentiviral vectors is stable expression of a transgene that has integrated into host genomic DNA, stem cells are considered a good target of lentiviral vectors. During myogenesis, activated-satellite cells or muscle progenitor cells proliferate in the muscle microenvironment. Most of these cells become differentiated and form myofibers, while small numbers of them are stored adjacent to myofibers as mitotically quiescent satellite cells for future muscle regeneration. Skeletal muscle of newborn mice may be a good target for lentiviral vectors, because of relatively high numbers of activated muscle progenitor cells contributing to muscle growth and satellite cell pools. Therefore, we tried targeting satellite cells or muscle progenitor cells using lentivirual vectors expressing dystrophin mini-gene.
We demonstrated that relatively higher levels of transuction were obtained with intra-muscular injection into neonatal muscles, and that both muscle fibers and primary cultured satellite cells stably expressed a GFP marker gene. We also demonstrated that lentiviral mediated truncated mini-dystrophin expression in mdx muscles may be useful for correction of pathological changes in dystrophic muscle.
These data suggest that myogenic stem cells can be stably transduced with lentiviral vectors and may contribute to stable muscle regeneration in dystrophic muscle by enabling continuous expression of mini-dystrophin, which may have implications for gene therapy of Duchenne muscular dystrophy.

12) Nucleofection and Phage phiC31 Integrase Mediate Stable Introduction of a Dystrophin Fusion Gene into Muscle Derived Stem Cell and Human Myoblasts

Simon P. Quenneville, Pierre Chapdelaine, Joel Rousseau, Nicolas J. Caron, Daniel Skuk, Eric C. Olivares, Michele P. Calos, Jacques P. Tremblay Human Genetic, CRCHUL, Sainte-Foy, QC, Canada; Department of Genetics, Stanford University School of Medicine, Stanford, CA

Ex vivo gene therapy offers a potential treatment for Duchenne muscular dystrophy by transfection of the dystrophin gene into the patient’s own myogenic precursor cells, followed by transplantation. This approach requires a safe procedure to stably modify myogenic cells so that they express the large dystrophin transgene. We used nucleofection to introduce DNA plasmids coding for eGFP or eGFP-dystrophin fusion protein and the phage phiC31 integrase into myogenic cells and to integrate these genes into a limited number of sites in the genome. This combination of methods eliminates the need for viral vectors and reduces the risk of insertional mutagenesis. Following nucleofection of a plasmid expressing eGFP, 50% of MD1 cells, a mouse muscle-derived stem cell line, and 60% of normal human primary cultured myoblasts transiently expressed the fluorescent protein. But stable expression was rare. In both cell types, co-nucleofection of a plasmid expressing the phiC31 integrase and a plasmid containing the eGFP gene carrying a 285 bp attB sequence produced 15 times more frequent stable eGFP expression, due to site-specific integration of the transgene into the genome. Co-nucleofection of the phiC31 integrase plasmid and of a large plasmid containing the attB sequence and the gene for an eGFP-full-length dystrophin fusion protein produced fluorescent human myoblasts that were able to form more intensely fluorescent myotubes after one month of culture. The presence of eGFP-full-length dystrophin protein in myotubes was confirmed by Western blotting. Finally, MD1 stem cells expressing integrated eGFP were successfully transplanted into leg muscles of mdx mice, leading to the presence of green fluorescent fibers. A non-viral approach combining nucleofection and the phiC31 integrase may eventually permit safe auto-transplantation of genetically modified myogenic cells to muscular dystrophy patients.

13) Successful AAV Vector-Mediated Gene Transfer into Canine Skeletal Muscle Required Suppression of Excess Immune Responses

Katsutoshi Yuasa, Madoka Yoshimura, Nobuyuki Urasawa, Katsujiro Sato, Yuko Miyagoe-Suzuki, John McC Howell, Shinichi Takeda Department of Molecular Therapy, National Institute of Neuroscience, NCNP, Kodaira, Tokyo, Japan; Division of Veterinary and Biomedical Sciences, Murdoch University, Perth, WA, Australia

Duchenne muscular dystrophy (DMD) is an X-linked, lethal muscle disorder caused by a mutation in the dystrophin gene (14 kb cDNA). An adeno-associated virus (AAV) vector-mediated gene transfer is one of attractive approaches to the treatment of DMD, but it has a limitation in insertion size up to 4.9 kb. To find a short but functional dystrophin cDNA, we have previously constructed three micro-dystrophin cDNAs, and generated transgenic (Tg) dystrophin-deficient mdx mice expressing micro-dystrophin. Among them, CS1-Tg mdx mice showed lowest levels of serum creatine kinase, complete amelioration of muscle pathology, and nearly full restoration of contractile force (BBRC. 293:1265, 2002). We also showed that muscle-specific MCK promoter in AAV vector could drive longer expression of the LacZ gene than the CMV promoter in skeletal muscle (Gene Ther. 23:1576, 2002). Furthermore, we constructed the AAV2 vector expressing ΔCS1 micro-dystrophin driven by MCK promoter, and demonstrated that AAV vector-mediated ΔCS1 transfer could ameliorate dystrophic phenotypes in mdx muscles (7th ASGT Annual Meeting 2004, in submission). For the application of this strategy to DMD patients, however, it is necessary to examine therapeutic effects and the safety issue in larger animal models, such as dystrophic dogs. We recently established a colony of beagle-based canine X-linked muscular dystrophy in Japan (Exp. Anim. 52: 93, 2003). When the AAV vector encoding the LacZ gene driven by a CMV promoter was introduced into skeletal muscles of dogs, β-galactosidase (β-gal) was expressed only in few fibers of injected muscle after 2 weeks of injection. No β-gal-positive fiver was detected in canine muscle at 4 and 8 weeks post-injection. Instead, large numbers of mononuclear cells appeared around β-gal-expressing fibers in injected muscle. To clarify mechanisms of low transduction and cellular infiltration in canine muscle after transfer of AAV vector, we examined viral infectivity, cytotoxicity and immune responses. First, we infected AAV vector into canine primary myotubes. This in vitro study showed that AAV vector could allow higher transgene expression in canine myotubes than in murine ones. Second, we tested whether injection of AAV particle elicit cytotoxicity or not. When the AAV vector expressing no transgene was injected into canine muscle, almost no infiltrating cells was observed in injected muscle. Third, we investigated immune responses. A lot of CD4- or CD8-positive cells were detected in clusters of infiltrating cells, together with elevated serum level of anti-β-gal IgG. To confirm low transduction depending on immune response, dogs received daily oral administration of cyclosporine (20 mg/kg/day) from -5 day of the introduction of the AAV vector. Immunosuppression considerably improved transduction efficiency by an AAV vector introduction in canine muscle. These results suggested that AAV vector-mediated gene transfer elicited stronger immune responses in canine muscle, and it was necessary to know the molecular background of excess immune responses and to find the way to minimize and suppress immune responses.

14) An AAV Vector-Mediated Micro-Dystrophin Expression in Relatively Small Percentage of Dystrophin-Deficient mdx Myofibers Still Improved the mdx Phenotype through Compensatory Hypertrophy

Madoka Ikemoto, Madoka Yoshimura, Miki Sakamoto, Yasushi Mochizuki, Katsutoshi Yuasa, Toshifumi Yokota, Yuko Miyagoe-Suzuki, Shin'ichi Takeda Department of Molecular Therapy, National Institute of Neuroscience,NCNP, Kodaira, Tokyo, Japan

[Background] Duchenne muscular dystrophy (DMD) is an X-linked, lethal muscle disorder caused by mutations in the dystrophin gene. An adeno-associated virus (AAV) vector-mediated gene transfer is one of attractive approaches to the treatment of DMD, though it has a limitation in insertion size up to 4.9 kb. Therefore, a full-length dystrophin cDNA (14 kb) cannot be incorporated into an AAV vector. We previously generated micro-dystrophin transgenic mdx mice. Micro-dystrophin CS1 transgenic mdx mice showed almost complete amelioration of dystrophic phenotypes (BBRC. 293: 1265, 2002).
[Objective] We constructed an AAV vector expressing micro-dystrophin ΔCS1, and introduced it into skeletal muscles of mdx mice and examined whether the dystrophic process had been ameliorated or not.
[Method] To incorporate micro-dystrophin CS1 cDNA (4.9 kb) into an AAV vector, we deleted 5’- and 3’-UTRs and exons 71-78 (alternative splicing regions), resulting 3.8 kb ΔCS1 cDNA. We produced type 2 AAV vector expressing ΔCS1 under the control of muscle specific MCK promoter to avoid immune response against transgene product (Gene Ther. 9: 1576, 2002), designated AAV2-MCKΔCS1. The vector was injected into anterior tibial (TA) muscles of 10-day-old and 5-week-old mdx mice. Mdx muscles show no obvious changes of degeneration at 10-day, whereas 5-week-old mdx muscles exhibit active cycles of muscle degeneration/regeneration.
[Result] When the AAV2-MCKΔCS1 was injected at 5 weeks of age, dystrophin-positive fibers were 51.5 ± 17.3% at 24 weeks after the injection. The ratio of centrally nucleated fibers in ΔCS1-positive fibers was significantly reduced compared with that of ΔCS1-negative fibers, indicating protective function of ΔCS1 against muscle degeneration. Furthermore, AAV-injected muscles revealed complete recovery of the specific tetanic force.
When injected at 10-day-old, ΔCS1-positive fibers was 16.5 ± 7.0% at 24 weeks. Most of ΔCS1-positive fibers were peripherally nucleated. Surprisingly, there was no statistical difference in specific tetanic force between AAV-injected mdx muscles and B10 muscles.
To clarify the mechanism of physiological recovery due to small numbers of ΔCS1-positive fibers, we examined the relationship between myofiber hypertrophy and force generation. We found positive correlation between the muscle weight and the force generation when injected at 10-day-old. To confirm whether increased muscle weight reflected myofiber hypertrophy, we measured cross section areas (CSAs) of individual fibers. Fiber CSAs were remarkably larger in ΔCS1-positive mdx fibers compared with ΔCS1-nagative, non-treated mdx fibers, and even B10 fibers. Thus, selective hypertrophy of ΔCS1-positive fibers seemed to greatly assist contractile force generation.
[Conclusion] The AAV2-MCKΔCS1 could be a powerful tool for the gene therapy of DMD. For clinical application of this strategy to DMD patients, experiments using a bigger animal model, e.g. canine X-linked muscular dystrophy will be very important.

15) Muscle-Derived Stem Cells Display an Extended, but Not Unlimited, Expansion Capability: Implication for Muscle Regeneration

Bridget M. Deasy, Burhan M. Gharaibeh, Michele Jones, Michael A. Lucas, Johnny Huard Bioengineering, University of Pittsburgh; Growth and Development Laboratory, Children's Hospital of Pittsburgh; Molecular Genetics & Biochemistry and Orthopedic Surgery, University of Pittsburgh Medical Center

Stem cells are frequently considered the optimal cell type for regenerative cell–based therapies; however they generally represent a small fraction of cells isolated from a biopsy or other cell source. Ex vivo cell expansion is a necessary step to obtain clinically relevant numbers of cells. In addition, stem cells are often theorized as cells with unlimited long-term expansion potential. The purpose of this study is to test the long-term expansion capability of a population of muscle-derived stem cells. We first examined the proliferation kinetics of murine muscle-derived stem cells (MDSCs) to determine if they obey Hayflick’s limit. We determined that these cells can be expanded for more than 300 population doublings (PDs) with no indications of replicative senescence. Next we examined how the molecular and behavioral stem cell phenotype, including the regenerative capacity, changes over time. We find that the MDSC population continues to maintain a relatively low level of desmin expression (<30%), and a high level of stem cell antigen 1 (Sca-1) expression (>65%) throughout the expansion. We observe that up to 200 PDs the MDSCs readily differentiate to form multinucleated myotubes, however expansion beyond 200 PDs leads to a decline in the number of cells entering the post-mitotic differentiated state. Remarkably, MDSC are capable of regenerating dystrophin expressing muscle fibers upon implantation in mdx muscular dystrophy model even after 200 population doublings. However, expansion beyond 200 PDs resulted in a subsequent decline in regeneration efficiency. Observed phenotypic changes highlight the inevitable aging of cells that results from cell expansion. Several findings including loss of contact inhibition, ability to grow on soft agar and an increase in numerical chromosomal abnormalities suggests that the MDSC may have become transformed. While the MDSC demonstrate a highly extended functional lifetime for muscle regeneration, we find that this potential is not unlimited.

16) Delivery of Igf-I and Dystrophin to Dystrophic mdx Muscle

Simone Abmayr, Paul Gregorevic, James M. Allen, Shanna M. Sawatzki, Jeffrey S. Chamberlain Department of Neurology, University of Washington School of Medicine, Seattle, WA

Duchenne muscular dystrophy is among the most common genetic diseases and is caused by mutations in the dystrophin gene. Dystrophic muscles display an extensive degeneration and regeneration process, whereby muscle fibers progressively lose their self-renewal potential and are gradually replaced by adipose and fibrotic tissue. Gene replacement therapy using truncated versions of dystrophin have been shown to protect dystrophic muscles from contraction-induced injury and partially reverse muscle pathology. An alternative approach involves the activation of satellite cells to maintain the regenerative potential of dystrophic muscle. Igf-I, an important mediator of cell growth and differentiation, has been shown to increase muscle mass and strength and to enhance muscle repair mechanism in dystrophic mdx muscles (Barton et al., 2002). However, Igf-1 is unable to restore mechanical integrity to muscle fibers lacking dystrophin. To determine if the beneficial effect of Igf-I is synergistic with the protective effect of dystrophin in ameliorating dystrophic pathology, we compared the effects of delivering Igf-I alone versus co-delivering both Igf-I and dystrophin to adult, dystrophic mdx mouse muscles. We generated recombinant adeno-associated viral vectors pseudotyped with the serotype 6 capsid protein that carry expression cassettes in which the muscle-specific creatine kinase promoter/enhancer drove either the micro-dystrophin (AAV-udys) or the Igf-I cDNA (AAV-Igf-I). Tibialis anterior muscles of mdx mice were injected with each vector separately, together or with buffer control and then analyzed four months post injection. Immunohistochemical analysis demonstrated persistent expression of dystrophin that reached an average of 40% of the total muscle cross sectional area. We also observed persistent expression of Igf-I mRNA at levels 200-400 fold greater than endogenous mdx Igf-I levels in AAV-Igf-I and AAV-udys co-injected muscles. In contrast, injection of AAV-Igf-I alone resulted in a 4-fold decline of Igf-I mRNA levels in the four months following injection into dystrophic mdx muscles. Functional measurements demonstrated that AAV-udys injected animals were partially protected from contraction-induced injury after two lengthening contractions, whereas animals injected with AAV-Igf-I alone were as susceptible as mdx animals to muscle damage. AAV-Igf-I treated animals, on the other hand, showed an increase in muscle mass, which was not seen after AAV-udys only treatment. In contrast, co-injection of AAV-Igf-I and AAV-udys resulted in increased muscle mass and muscle strength, and in protection from contraction-induced injury. These results suggest that the combination of AAV-Igf-I and AAV-udys acted synergistically and was beneficial for the animal.

17) Immunogenicity of Dystrophins Delivered to Mice by Gutted Adenoviral Vectors

Jie Mi, Leonard Leuse, Jeannine Scott, Shannon Barker, Shanna Sawatzki, Dennis Hartigan-O'Connor, Jeffrey S. Chamberlain Neurology, University of Washington, School of Medicine, Seattle, WA; Etubics Corporation, Ellensburg, WA

Duchenne muscular dystrophy (DMD) is an X-linked recessive genetic disorder resulting from mutations in the dystrophin gene. Delivery of a therapeutic dystrophin gene back into the diseased muscle has been suggested as a means of gene therapy for DMD. Previous work in this lab has demonstrated that using a gutted adenovirus vector (gAd), a full-length dystrophin cDNA expression cassette can be delivered into muscles of the mdx (dystrophin-deficient) mouse, resulting in efficient transduction of myofibers with dystrophin. A long-term follow-up study has revealed that the gAd delivered, muscle-specific promoter-driven, gene expression could be sustained for up to six months in adult mdx mice when delivering mouse dystrophin or mouse utrophin. In contrast, delivery of full-length human dystrophin (hDys) using gAd vectors resulted in a slow loss of gene expression over the 6-month window. Furthermore, an increase in the number of infiltrating CD4+ and CD8+ T-cells was observed in muscles injected with the human, but not the mouse, dystrophin-expressing gAd vectors. These results suggest that a host immune response has been elicited, albeit possibly mild, against the hDys-expressing muscle fibers. Despite this slow loss of hDys expression, a number of small caliber, regenerating myofibers were observed at the 6 month time point that expressed the human protein, suggesting that gAd vectors can be retained for many months in satellite cells even in the presence of an immune response against dystrophin expression in myofibers.
Other vector systems with a more limited cloning capacity, including adeno-associated viral (AAV) vectors, have also been tested for delivery of a variety of truncated dystrophin genes into mdx muscles, to test the hypothesis that specific domains within the dystrophin protein could be sufficient to achieve a functional recovery of muscle. Additional knowledge of the location of immunogenic epitopes in the dystrophin protein could facilitate the design of optimally truncated dystrophin mini-genes that would minimize the potential for an immune response against exogenous dystrophin.
We hypothesize that the immuonogenicity of the dystrophin protein varies depending on 1) the mode of delivery, e.g. gAd-vectored vs. AAV-vectored vs. naked DNA injection; 2) the route of administration, e.g. intramuscular vs. intravenous injection; or 3) the expression pattern, e.g. muscle-restricted expression vs. ubiquitous expression. We are testing cellular immune responses against three different domains of the dystrophin protein, following intramuscular delivery by gutted adenovirus vectors. Furthermore, the immune responses to these three domains are also being compared between systemically delivered dystrophin vs. intramuscularly delivered dystrophin.

18) Human Myoblast Deimmortalization Using Tat-Mediated Cre Recombinase Delivery

Nicolas J. Caron, Marie-Eve Ducharme, Philippe Mills, Simon P. Quenneville, Jacques P. Tremblay Human Genetics, CHUL Research Center, Quebec City, QC, Canada

The reduced proliferating capacity of myoblasts isolated from Duchenne muscular dystrophy (DMD) patients limits our capacity to genetically modify and proliferate them in vitro for their use in autologous transplantation. Previously, our research group has successfully immortalized and extensively proliferated DMD myoblasts using the SV-40 Large T antigen (TAg) and hTERT ^1,2. Using a retroviral vector coding for TAg flanked by LoxP sites, immortalization reversal can be performed by Cre delivery. To circumvent the requirement for an additional infection, we used a Tat-Cre recombinase fusion protein to exert the recombination necessary for immortalization reversal. Tat-Cre intracellular transduction produced site-specific recombination and excision of the TAg. To facilitate the TAg excision process, cell lines containing a single immortalizing integrative event were generated. Using these cell lines, we demonstrate that by optimizing the delivery using a high concentration Tat-Cre protein, by co-incubating with chloroquine and by selecting against cells containing copies of the unrecombined vector, complete TAg removal could be achieved with a single 1 h treatment. In addition to the molecular evidence demonstrating immortalizing cassette removal, TAg excision also resulted in growth arrest within 2 days. The resulting cell culture could be maintained for at least 2 weeks. These results indicate that the intracellular delivery of a recombinant Tat-Cre protein could be a useful tool for a variety of applications that necessitate the manipulation of cells in culture.
1. Seigneurin-Venin, S., Bernard, V. & Tremblay, J.P. Telomerase allows the immortalization of T antigen-positive DMD myoblasts: a new source of cells for gene transfer application. Gene Ther 7, 619-23 (2000).
2. Seigneurin-Venin, S. et al. Transplantation of normal and DMD myoblasts expressing the telomerase gene in SCID mice. Biochem Biophys Res Commun 272, 362-9 (2000).

19) The Fetal Approach: A Novel Therapy for the Treatment of Musculo-Skeletal Disease

Michael Themis, Lisa G. Gregory, Simon N. Waddington, Maxine V. Holder, Kyriacos A. Mitrophanous, Suzanne M. K. Buckley, Brian W. Bigger, Fiona E. Ellard, Lucy E. Walmsley, Pippa Radcliffe, Nick Mazarakis, Mimoun Azzouz, Lorraine Lawrence, Terrence Cook, Faisal A. Allaf, Susan Kingsman, Charles Coutelle Ceel and Molecular Biology, Imperial College, London, United Kingdom; OxfordBiomedica, Oxford, United Kingdom

Gene therapy for Duchenne muscular dystrophy has so far not been successful because of the difficulty in achieving efficient and permanent gene transfer to the large number of affected muscles and the development of immune reactions against vector and transgenic protein. In addition, the prenatal onset of disease complicates postnatal gene therapy. We have therefore proposed a fetal approach to overcome these barriers. We have applied b-galactosidase expressing EIAV lentiviruses by single or combined injection via different routes to the MF1 mouse fetus on day 15 of gestation and describe substantial gene delivery to the musculature. Highly efficient gene transfer to skeletal muscles, including the diaphragm and intercostal muscles, as well as to cardiac myocytes was observed and gene expression persisted for at least five months after administration of this integrating vector. Using alternative envelope glycoproteins to pseudotype the EIAV vector also appears to provide improved gene targeting not only to muscle fibres but also muscle satellite cells important for muscle regeneration. These findings support the concept of in utero gene delivery for therapeutic and long term prevention/correction of muscular dystrophies and pave the way for a future application in the clinic.