Hironori Okada

Long-term Engraftment of Multipotent Mesenchymal Stromal Cells That Differentiate to Form Myogenic Cells in Dogs With Duchenne Muscular Dystrophy

Duchenne muscular dystrophy (DMD) is an incurable genetic disease with early mortality. Multipotent mesenchymal stromal cells (MSCs) are of interest because of their ability to differentiate to form myogenic cells in situ. In the present study, methods were developed to expand cultures of MSCs and to promote the myogenic differentiation of these cells, which were then used in a new approach for the treatment of DMD. MSC cultures enriched in CD271(+) cells grew better than CD271-depleted cultures. The transduction of CD271(+) MSCs with MyoD caused myogenic differentiation in vitro and the formation of myotubes expressing late myogenic markers. CD271(+) MSCs in the myogenic cell lineage transplanted into dog leukocyte antigen (DLA)-identical dogs formed clusters of muscle-like tissue. Intra-arterial injection of the CD271(+) MSCs resulted in engraftment at the site of the cardiotoxin (CTX)-injured muscle. Dogs affected by X-linked muscular dystrophy in Japan (CXMD(J)) treated with an intramuscular injection of CD271(+) MSCs similarly developed muscle-like tissue within 8-12 weeks in the absence of immunosuppression. In the newly formed tissues, developmental myosin heavy chain (dMyHC) and dystrophin were upregulated. These findings demonstrate that a cell transplantation strategy using CD271(+) MSCs may offer a promising treatment approach for patients with DMD.

Improvement of cardiac fibrosis in dystrophic mice by rAAV9-mediated microdystrophin transduction.

Duchenne muscular dystrophy (DMD) is the most common form of the progressive muscular dystrophies characterized by defects of the dystrophin gene. Although primarily characterized by degeneration of the limb muscles, cardiomyopathy is a major cause of death. Therefore, the development of curative modalities such as gene therapy is imperative. We evaluated the cardiomyopathic features of mdx mice to observe improvements in response to intravenous administration of recombinant adeno-associated virus (AAV) type 9 encoding microdystrophin. The myocardium was extensively transduced with microdystrophin to significantly prevent the development of fibrosis, and expression persisted for the duration of the study. Intraventricular conduction patterns, such as the QRS complex duration and S/R ratio in electrocardiography, were also corrected, indicating that the transduced microdystrophin has a protective effect on the dystrophin-deficient myocardium. Furthermore, BNP and ANP levels were reduced to normal, suggesting the absence of cardiac dysfunction. In aged mice, prevention of ectopic beats as well as echocardiographic amelioration was also demonstrated with improved exercise performance. These findings indicate that AAV-mediated cardiac transduction with microdystrophin might be a promising therapeutic strategy for the treatment of dystrophin-deficient cardiomyopathy.

Dystrophic mdx mice develop severe cardiac and respiratory dysfunction following genetic ablation of the anti-inflammatory cytokine IL-10

Duchenne muscular dystrophy (DMD) is a progressive muscle-wasting disease that causes respiratory and cardiac failure. Inflammation is a key pathological characteristic of dystrophic muscle lesion formation, but its role and regulation in the disease time course has not been sufficiently examined. In the present study, we used IL-10(-/-)/mdx mice lacking both dystrophin and the anti-inflammatory cytokine, interleukin-10 (IL-10), to investigate whether a predisposition to inflammation affects the severity of DMD with advancing age. The IL-10 deficiency caused a profound DMD phenotype in the dystrophic heart such as muscle degeneration and extensive myofiber loss, but the limb muscle and diaphragm morphology of IL-10(-/) (-)/mdx mice was similar to that of mdx mice. Extensive infiltrates of pro-inflammatory M1 macrophages in regeneration of cardiotoxin-injured muscle, altered M1/M2 macrophage phenotype and increased pro-inflammatory cytokines/chemokines production were observed in the diaphragm and heart of IL-10(-/-)/mdx mice. We characterized the IL-10(-/-)/mdx mice as a dystrophic model with chronic inflammation and severe cardiorespiratory dysfunction, as evidenced by decreased percent fractional shortening (%FS) and ejection fraction percent (EF%) on echocardiography, reduced lower tidal volume on whole-body plethysmography. This study suggests that a predisposition to inflammation is an important indicator of DMD disease progression. Therefore, the development of anti-inflammatory strategies may help in slowing down the cardiorespiratory dysfunction on DMD.

Intra-Amniotic rAAV-Mediated Microdystrophin Gene Transfer Improves Canine X-Linked Muscular Dystrophy and May Induce Immune Tolerance

Duchenne muscular dystrophy (DMD) is a severe congenital disease due to mutations in the dystrophin gene. Supplementation of dystrophin using recombinant adenoassociated virus vector has promise as a treatment of DMD, although therapeutic benefit of the truncated dystrophin still remains to be elucidated. Besides, host immune responses against the vector as well as transgene products have been denoted in the clinical gene therapy studies. Here, we transduced dystrophic dogs fetuses to investigate the therapeutic effects of an AAV vector expressing microdystrophin under conditions of immune tolerance. rAAV-CMV-microdystrophin and a rAAV-CAG-luciferase were injected into the amniotic fluid surrounding fetuses. We also reinjected rAAV9-CMV-microdystrophin into the jugular vein of an infant dystrophic dog to induce systemic expression of microdystrophin. Gait and cardiac function significantly improved in the rAAV-microdystrophin-injected dystrophic dog, suggesting that an adequate treatment of rAAV-microdystrophin with immune modulation induces successful long-term transgene expression to analyze improved dystrophic phenotype.

Ultracentrifugation-free chromatography-mediated large-scale purification of recombinant adeno-associated virus serotype 1 (rAAV1).

Recombinant adeno-associated virus (rAAV) is an attractive tool for gene transfer and shows potential for use in human gene therapies. The current methods for the production and purification of rAAV from the transfected cell lysate are mainly based on cesium chloride and iodixanol density ultracentrifugation, although those are not scalable. Meanwhile, chromatography-based systems are more scalable. Therefore, in this study, we developed a novel method for the production and purification of rAAV serotype 1 (rAAV1) from serum-free culture supernatant based on ion-exchange and gel-filtration chromatography to obtain highly purified products with an ultracentrifugation-free technique towards Good Manufacturing Practice (GMP) production. The purified rAAV1 displayed three clear and sharp bands (VP1, VP2, and VP3) following sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and more than 90% of rAAV1 particles contained fully packaged viral genomes according to negative-stain electron micrographic analysis. Consequently, the resultant genomic titer of the purified rAAV1 was 3.63 × 1013 v.g./ml (the total titer was 4.17 × 1013 v.g.) from the 4 × 109 HEK293 cells. This novel chromatography-based method will facilitate scale-up of manufacturing for clinical applications in gene therapy.

Ultrasound-guided non-surgical embryo collection in the common marmoset

Experimental primate embryology has been hampered by limited access to embryos. In addition to surgical techniques, the less stressful non-surgical technique of uterine flushing has been developed but has had only limitedly used in recovering pre-implantation embryos from marmoset monkeys. In this study, we introduce the use of ultrasonography during marmoset non-surgical uterine flushing to make the cannulation easier, to further reduce stress, and to ensure thorough uterine flushing. We were able to cannulate in 99% of the transcervical cannulation attempts, repeat the flushing up to 17 times with the same animal, and recover up to 90% of the ovulation products. We also found that 8-cell or earlier stage embryos could be frequently obtained by non-surgical uterine flushing at 4 or 5 days after ovulation. The easiness and effectiveness of this novel ultrasound-guided technique will enable more research groups to study marmoset embryology and facilitate progress in this field.

Efficient embryo transfer in the common marmoset monkey (Callithrix jacchus) with a reduced transfer volume: a non-surgical approach with cryopreserved late-stage embryos.

ABSTRACT Among primates, the common marmoset is suitable for primate embryology research. Its small body size, however, has delayed the technical development of efficient embryo transfer. Furthermore, three factors have been determined to adversely affect the performance of marmoset embryo transfer: nonsurgical approaches, the use of cryopreserved embryos, and the use of late-stage embryos. Here we performed embryo transfer under conditions that included the above three factors and using either a small (1 μl or less) or a large volume (2–3 μl) of medium. The pregnancy and birth rates were 50% (5/10) and 27% (3/11), respectively, when using the large volume, and 80% (8/10) and 75% (9/12), respectively, when using the small volume. The latter scores exceed those of previous reports using comparable conditions. Thus, it appears that these three previously considered factors could be overcome, and we propose that reducing the transfer volume to 1 μl or less is essential for successful marmoset embryo transfer.

Robust Long-term Transduction of Common Marmoset Neuromuscular Tissue With rAAV1 and rAAV9

Profiles of recombinant adeno-associated virus (rAAV)-mediated transduction show interspecies differences for each AAV serotype. Robust long-term transgene expression is generally observed in rodents, whereas insufficient transduction is seen in animals with more advanced immune systems. Non-human primates, including the common marmoset, could provide appropriate models for neuromuscular diseases because of their higher brain functions and physiological resemblance to humans. Strategies to induce pathologies in the neuromuscular tissues of non-human primates by rAAV-mediated transduction are promising; however, transgene expression patterns with rAAV transduction have not been elucidated in marmosets. In this study, transduction of adult marmoset skeletal muscle with rAAV9 led to robust and persistent enhanced green fluorescent protein (EGFP) expression that was independent of the muscle fiber type, although lymphocyte infiltration was recognized. Systemic rAAV injection into pregnant marmosets led to transplacental fetal transduction. Surprisingly, the intraperitoneal injection of rAAV1 and rAAV9 into the neonatal marmoset resulted in systemic transduction and persistent transgene expression without lymphocyte infiltration. Skeletal and cardiac muscle were effectively transduced with rAAV1 and rAAV9, respectively. Interestingly, rAAV9 transduction led to intense EGFP signaling in the axons of the corpus callosum. These transduction protocols with rAAV will be useful for investigating gene functions in the neuromuscular tissues and developing gene therapy strategies.

Effect of the size of zona pellucida opening on hatching in the common marmoset monkey (Callithrix jacchus) embryo

The use of the common marmoset monkey in biomedical research has increased recently, and further attention has been devoted to this model after the successful production of transgenic marmosets. To extend genetic engineering approaches to widespread biomedical research fields, efficient prolonged in vitro culturing of embryo development is necessary. We aimed to evaluate the effects of the size of the zona pellucida opening on promoting the hatching process in the marmoset embryo. Piezo-microdrilling of a 6-μm opening in eight embryos resulted in four partially hatched embryos and one hatched embryo after 5 days of culture. Piezo-microdrilling a 20-μm opening in 11 embryos resulted in nine partial hatchings and no hatched embryos. Piezo-scraping an 80-μm opening in six embryos resulted in no partially hatched embryos and five hatched embryos. These results suggest that an 80-μm opening, rather than 6-μm or 20-μm openings, is suitable to complete the hatching process in the marmoset embryo.

308. Ultracentrifugation-Free Chromatography-Mediated Large-Scale Purification of Recombinant Adeno-Associated Virus Serotype 1 (rAAV1) and rAAV9 from the Serum-Free Culture Supernatant

[Background]The current production of rAAV from the transfected cell lysate and purification based on CsCl or iodixanol density ultracentrifugation are not suitable for large-scale processing. Although rAAV1 and rAAV9 are promising therapeutic vectors for genetic neuromuscular disorders, the large-scale purification method for those vectors has not yet been established. In this study, we elaborate the novel chromatography-mediated methods for purification of rAAV1 and rAAV9 from the serum-free culture supernatant with ultracentrifugation-free technique towards large-scale and GMP production.[Methods]rAAV1 (scAAV1-CBA-EGFP) and rAAV9 (scAAV9-CBA-EGFP) were produced by the triple-transfection to HEK293 or HEK293EB cells in serum-free medium with polyethyleneimine (PEI). Five days later, the culture supernatant was tangential flow-filtrated (TFF) and concentrated by the Hollow Fiber system. After reducing protein debris by 1/3-saturated ammonium sulfate (1/3 AS) precipitation, rAAV1 or rAAV9 was precipitated in 1/2 AS solution.Subsequently, the precipitated rAAV1 was dissolved in 10 ml of PBS containing 3 mM MgCl2. After the sample was dialyzed against a buffer containing 5 mM NaCl, the dialysate was diluted with dH2O and loaded to tandem quintet cation-exchange membranes (Mustang SXT) and quintet anion-exchange membranes (Mustang QXT) with a column volume of 0.86 ml. After rAAV1 was eluted from Mustang QXT, it was finally purified by gel-filtrated chromatography (Superdex 200 10/300 GL).The precipitated rAAV9 was dissolved in 22 ml of 3.3 mM MES, 3.3 mM HEPES, 3.3 mM sodium acetate (MHN) buffer (pH8.0) containing 50 mM NaCl and 0.01% Pluronic F-68. After the resultant sample was diluted with MHN buffer, it was loaded to tertiary amine charged anion-exchange column. The passed through fraction containing rAAV9 was finally purified by gel-filtrated chromatography.The physiological and biological properties of the purified rAAV1 and rAAV9 were characterized by qPCR, electron micrograph, FACS, western blot and SDS-PAGE.[Results]The purified rAAV1 and rAAV9 displayed three major bands (VP1, VP2, and VP3) on SDS-PAGE and more than 90% of rAAV1 particles or 95% of rAAV9 particles was contained fully packaged viral genomes according to electron micrographic analysis. We confirmed increasing infectivity of rAAV1 products depending on chromatographic purification step and that of rAAV9 is under evaluation. The resultant genomic titer of the purified rAAV1 was 4.17 × 1013 v.g. (3.63 × 1013 v.g./ml) from the 4 × 109 HEK293 cells and the purified rAAV9 was 1.49 × 1015 v.g. (1.14 × 1014 v.g./ml) from the 5.1 × 109 HEK293EB cells.[Conclusion]Chromatography-mediated purification from the culture supernatant is a major breakthrough, especially for the production of rAAV9. We obtained rAAV1 and rAAV9 by this protocol with high titer, high purity and minimum contamination of empty particles. This novel chromatography-based method would be consistent with GMP production and facilitate clinical studies in the future.