Hiromi Hayashita-Kinoh

Scalable Purification of Adeno-associated Virus Serotype 1 (AAV1) and AAV8 Vectors, Using Dual Ion-Exchange Adsorptive Membranes

In vivo gene transduction with adeno-associated virus (AAV)-based vectors depends on laborious procedures for the production of high-titer vector stocks. Purification steps for efficient clearance of impurities such as host cell proteins and empty vector particles are required to meet end-product specifications. Therefore, the development of alternative, realistic methods to facilitate a scalable virus recovery procedure is critical to promote in vivo investigations. However, the conventional purification procedure with resin-based packed-bed chromatography suffers from a number of limitations, including variations in pressure, slow pore diffusion, and large bed volumes. Here we have employed disposable high-performance anion- and cation-exchange membrane adsorbers to effectively purify recombinant viruses. As a result of isoelectric focusing analysis, the isoelectric point of empty particles was found to be significantly higher than that of packaged virions. Therefore, AAV vector purification with the membrane adsorbers was successful and allowed higher levels of gene transfer in vivo without remarkable signs of toxicity or inflammation. Electron microscopy of the AAV vector stocks obtained revealed highly purified virions with as few as 0.8% empty particles. Furthermore, the membrane adsorbers enabled recovery of AAV vectors in the transduced culture supernatant. Also, the ion-exchange enrichment of retroviral vectors bearing the amphotropic envelope was successful. This rapid and scalable viral purification protocol using disposable membrane adsorbers is particularly promising for in vivo experimentation and clinical investigations.

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.

Alteration in the differentiation-related molecular expression in the subventricular zone in a mouse model of Parkinson's disease

Enhancement of neurogenesis could be a suitable treatment approach to up-regulate dopaminergic neurons in Parkinsons disease (PD). In the present study, we focused on the kinetics of the subventricular zone (SVZ) in a mouse model of PD induced by MPTP injection. We showed recently the proliferation potential of neuronal stem cells (NSCs) prepared from the olfactory bulb of an animal model of PD [Hayakawa, H., Hayashita-Kinoh, H., Nihira, T., Seki, T., Mizuno, Y., Mochizuki, H., 2007. The isolation of neural stem cells from the OB of Parkinsons disease model. Neurosci. Res.]. In this study, we examined the relationship between proliferation and differentiation of NSCs in SVZ of both acute and chronic PD models. Only acute MPTP treatment significantly increased the areas of glial fibrillary acidic protein (GFAP)-expressing cells and decreased the areas of polysialylated neural cell adhesion molecule (PSA-NCAM)-expressing cells in the SVZ. In the case of caspase-11 knockout mice, MPTP did not induce alteration in the areas of GFAP-expressing cells and PSA-NCAM-expressing cells. Our results suggest that neuroinflammation related to the caspase-11 cascade in the striatum regulates differentiation of neural stem cells in the SVZ of our mouse model of PD.

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.

The isolation of neural stem cells from the olfactory bulb of Parkinson's disease model

Hyposmia is one of the characteristic symptoms of PD. We isolated the neurosphere forming cells (NSFCs) from the olfactory bulb (OB) after dopaminergic neuronal loss induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which is a model of Parkinsons disease. We used BrdU to label dividing cells and isolated NSFCs from the OB of adult mice with or without MPTP to confirm the function of OB in PD models. Seven days after MPTP treatment, BrdU-positive cells were significantly increased in the OB, especially in the glomerular layer (GL) and the subependymal zone (SEZ). The number of neurospheres derived from the adult OB was not decreased in groups receiving MPTP, instead, it was significantly increased at 21 days post-injection and only returned to control levels 40 days after MPTP administration. We also evaluated the differentiation of NSFCs into neural subtypes and found that these NSFCs could be well infected with retrovirus. Adult neurogenesis may be enhanced as a repair system in the tyrosine hydroxylase (TH) positive cells of the OB after MPTP administration. The isolation of neural stem cells from the OB after MPTP administration has helped to establish the cellular basis of neurogenesis and supports a role for the transplant-mediated treatment of PD.

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.

337. Transient Ultrasound-Mediated Microbubble-Assisted Modulation of Blood-Brain Interface in Adult Common Marmoset to Improve rAAV-Mediated Brain Transduction

Background: Non-human primates (NHPs) could provide an appropriate model for neuromuscular diseases because of its cognitive function and physiological resemblance to human. Production of transgenic and knockout NHPs from preimplantation embryos by using genomic modification were reported. However, several decades would be required for establishing homozygous and/or phenotypically stable progenies and for developing the symptoms in case of late onset diseases. In contrast, induction of pathology with recombinant adeno-associated virus (rAAV) has potential to break through this situation, because it is possible to realize with the existing aged animals. In this context, fully maturated blood-brain interface (BBI) significantly limits passive rAAV transport from circulation to the brain. To overcome this issue, we investigated BBI opening as a promising technology to make cerebral capillary open transiently by resonance of i.v. injected microbubble (MB) locally excited with ultrasound irradiation (UI).Methods: Evans blue (EB) and aminoisobutyric acid (AIB) were used for a tracer that has high affinity for serum albumin (ALB). MB and EB were i.v. injected as bolus into the femoral vein of the anesthetized adult marmosets. Transcranial (TC) UI to the brain was performed for 5 minutes, and the brain was examined one day after the UI. To verify the leakage, cryosections were immunostained against ALB and CD31. For live imaging analysis, MB, carbon 11-labeled AIB, and rAAV1 were i.v. injected into the tail vein. Subsequently, TCUI and PET scan were performed. At two days after TCUI, rAAV1 with distinguishable another vector genome was i.v. injected again. At one week after TCUI, the brain was sampled, and pieced into various parts. Then relative rAAV genome copies were measured by qPCR.Results: Macroscopically, ALB leakage was recognized in temporal cortex and hippocampus when TCUI was performed towards the temporal region. When TCUI was performed towards all direction, it was found in whole brain except for white matter. Microscopically, the blood vessels of 10-50 µm in diameter especially in the hippocampus were sensitive to BBI opening. From live image, leakage in the hippocampus, anterior cingulate cortex, basal ganglia, optic tract, amygdala, and superior temporal gyrus started just after TCUI. Lower genome copies of the secondary injected rAAV1 at two days after TCUI revealed that the BBI opening was transient.Conclusion: UI-mediated MB-assisted TC-BBI opening is a promising approach to generate the disease model based on adult common marmoset.

378. Improved Transduction of Canine X-Linked Muscular Dystrophy with rAAV9-Microdystrophin by Introducing Immune Tolerance

Background: Duchenne muscular dystrophy (DMD) is a congenital disease causing progressive deterioration of skeletal and cardiac muscles because of mutations in the dystrophin gene. Supplementation of dystrophin using rAAV is effective to improve pathogenesis of animal models of DMD. However, we have previously reported that local injection of rAAV2 or rAAV8 to canine skeletal muscles without immunosuppression resulted in insufficient transgene expression with potent immune responses. Here we used DMD dog (CXMDJ) to investigate three strategies of inducing immune tolerance to the rAAV vector and transgene expression with rAAV9-microdystrophin (rAAV-µDys).Methods: For fetal transduction, we tried two methods to induce immune tolerance against rAAV and microdystrophin. First, direct injection of rAAV into amniotic fluid at embryonic day 35 (oral ingestion of rAAV). Second, pregnant CXMDJ heterozygote with embryonic day 30 fetuses was injected with rAAV by intravenous injection (trans-placental rAAV transduction). Furthermore, for postnatal transduction, we tried mesenchymal stem cells (MSCs) pretreatment with rAAV transduction. Bone-marrow derived MSCs and rAAV9-Luciferase or rAAV9-µDys were intramuscularly or intravenously injected into the normal or CXMDJ dog at 8 weeks old. Seven days after injection, MSCs were systemically injected again. At 8 days after 1st injection, rAAV9-Luciferase or rAAV9-µDys was intramuscularly or intravenously injected into the same dog. To examine the immune response against rAAV, purified canine peripheral leukocytes were exposed to rAAV9 for 4 hours, and then IFN-γ expression was analyzed using qRT-PCR. Skeletal muscles of the rAAV-Luc or rAAV-µDys injected animals were sampled by biopsy for expression analysis at 4 weeks after rAAV injection.Results: Following the fetal transduction, expression of IFN-γ in the purified peripheral blood leukocytes after the rAAV exposure were not induced in both of the rAAV oral ingestion and trans-placental transduced dogs, suggesting the successful induction of immune tolerance against rAAV. rAAV-derived microdystrophin expression were confirmed by immunohistochemistry in the transduced affected dogs from additional rAAV injection in both methods. In normal or CXMDJ puppy, administration of rAAV-Luc or rAAV-µDys following MSCs treatment resulted in higher expression of transgene, compared to the rAAV transduction alone. Expression of IFNγ in the purified peripheral blood leukocytes after the rAAV exposure were not enhanced in the rAAV with MSCs, suggesting the immune suppressive effects of the MSCs.Conclusion: Our results demonstrate that induction of immune tolerance against rAAV and/or transgene can be achieved both by fetal or postnatal rAAV injections. These strategies would be effective approach to analyze the expression and function of transgene in vivo. These findings also support the future feasibilities of rAAV-mediated protein supplementation strategies.